Use of inhibitors of leukotriene B4 receptor BLT2 for treating asthma

ABSTRACT

The present invention relates to the use of inhibitors of leukotriene B4 receptor BLT2 for treating asthma. More particularly, the present invention relates to a pharmaceutical composition for treating asthma comprising BLT2 inhibitors and a method for treating asthma using BLT2 inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/316,015, filed onDec. 9, 2011 and published as U.S. Patent Application Publication No.2012-0164149. U.S. Ser. No. 13/316,015 is a continuation-in-part of U.S.application Ser. No. 12/450,342, filed on Sep. 22, 2009, and publishedas U.S. Patent Application Publication No. 2010/0034835, which is theU.S. national phase, pursuant to 35 U.S.C. §371, of InternationalApplication No. PCT/KR2008/001650, which was filed on Mar. 24, 2008, andpublished as International Publication No. WO 2008/117971, which claimsthe benefit of U.S. Provisional Application No. 60/896,501, which wasfiled on Mar. 23, 2007, and 60/896,502, which was filed on Mar. 23,2007, the disclosures of which are hereby incorporated in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 6, 2014, isnamed 8312DIV250498_ST25.txt and is 33,498 bytes in size.

FIELD OF INVENTION

The present invention relates to use of inhibitors of leukotriene B4receptor BLT2 for treating asthma. More particularly, the presentinvention relates to a pharmaceutical composition for treating asthmacomprising BLT2 inhibitors and a method for treating asthma using BLT2inhibitors.

BACKGROUND OF THE INVENTION

Leukotriene B4 (LTB₄) is a key mediator of inflammatory processes,immune responses, and host defenses against infection (1-4). Itstimulates chemotaxis, degranulation, release of lysosomal enzymes, andthe production of reactive oxygen species (ROS) (5-7). In fact, LTB₄ isone of the most potent chemoattractants known, acting mainly ongranulocytes and monocytes (8, 9). Recently, it was also shown to be achemoattractant for effector CD4+ and CD8+ T lymphocytes, recruitingthem to sites of acute inflammation (10-15). It also promotes celladhesion to vascular endothelial cells and transmigration (8, 16), whichamplifies inflammatory early responses. Although LTB₄-induced leukocyterecruitment is thought to play a protective role in host defense againstvarious pathogens, it is also involved in a number of human inflammatorydiseases such as asthma (17-20), a disease of chronic airwayinflammation characterized by eosinophilic infiltration, mucushypersecretion, and airway hyperresponsiveness (AHR). Thus, asignificantly increased level of LTB₄ is detected in the airways ofpatients with asthma and also in experimental models of asthma (20).

LTB₄ produces its biological effects via specific G protein-coupledreceptors known as BLT1 and BLT2 (21-24). To date, most studies of LTB₄receptors have focused on the high-affinity LTB₄ receptor, BLT1,expressed exclusively in leukocytes, especially its role in inflammatoryresponses (22). For example, early recruitment of neutrophils andeosinophils into the airways in response to allergen inhalation isreduced in BLT1-deficient mice (8, 25), suggesting a role of BLT1 in thechemotaxis of granulocytes in allergic asthma. In addition, BLT1 isessential for the allergen-mediated early recruitment of CD4+ and CD8+ Tcells into the lung airways and the development of allergen-induced AHRand inflammation under certain experimental conditions (26, 27). Incontrast to BLT1, BLT2 has a low affinity for LTB₄ and is expressed in awide variety of tissues, with highest levels in the spleen, leukocytesand ovary (23).

The role of BLT2 in the pathogenesis of asthma was investigated using amurine model. By employing antisense to block endogenous BLT2expression, a critical role for BLT2 in the development of AHR andairway inflammation was demonstrated. In addition, without being boundto a particular theory, BLT2 causes asthmatic symptoms by elevating ROSgeneration and subsequent NF-KB activation. Furthermore,immunohistochmical analysis of clinical asthma samples revealed asignificant elevation of expression of BLT2 mainly in the airwayepithelial layers as well as in the microvascular endothelium, which issimilar to the pattern observed in the murine model of asthma.

Asthma is thought to be caused by a combination of genetic andenvironmental factors. The prevalence of asthma has increasedsignificantly since the 1970s. As of 2010, 300 million people wereaffected worldwide. In 2009 asthma caused 250,000 deaths globally.Symptoms of asthma can be prevented by avoiding triggers, such asallergens and irritants, and by inhaling corticosteroids. However,long-term corticosteroid use has the potential to cause severeside-effects including, e.g., hyperglycemia, insulin resistance,diabetes mellitus, osteoporosis, cataract, anxiety, depression, colitis,hypertension, ictus, erectile dysfunction, hypogonadism, hypothyroidism,amenorrhoea, and retinopathy. Accordingly, new compositions and methodsfor treating asthma are urgently required.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide use of aBLT2 inhibitor (e.g., an antibody against long form BLT2) in amedicament for the treatment of asthma.

Further, another object of the present invention is to provide apharmaceutical composition for the treatment of asthma comprising a BLT2inhibitor (e.g., an antibody against long form BLT2) as an activeingredient.

Further, another object of the present invention is to provide a methodfor treating a patient with asthma, which comprises administering aneffective amount of BLT2 inhibitor (e.g., an antibody against long formBLT2) to the patient.

Further, another object of the present invention is to provide a methodfor screening a substance for treating asthma, which comprisesdetermining whether to reduce the expression or signaling level of BLT2.

Further, another object of the present invention is to provide a kit fordetecting asthma, which comprises a primer or probe for detecting a BLT2nucleic acid molecule or an antibody for detecting BLT2 protein.

Further, another object of the present invention is to provide use of aRac inhibitor in a 25 medicament for the treatment of asthma.

Thus, the invention provides use of BLT2 inhibitors (e.g., anti-longform BLT2 antibody) for (1) suppressing an allergic response (e.g.,asthma), (2) suppressing immune responses of mast cells, (3) suppressingTh2 cytokine IL-13 release, and (4) suppressing one or more ofeosinophil infiltration into lung airway, airway inflammation, andairway hyperresponsiveness. Also, this 30 invention include (4) a novelstrategy for screeing BLT2 signaling inhibitors by measuring the cellgrowth of Rat2-BLT2 stable cells. Thus, the invention claims the use ofstrategies targeting BLT2 overexpression or over-activation as a toolfor developing therapeutic composition against asthma.

The invention is at least based in part on the discovery of arelationship between asthma and a BLT2-linked signaling cascade. Withoutbeing bound to a particular theory, LTB₄ exerts its effects through bothBLT1- and BLT2-dependent signaling pathways and that the two maycooperate during the development of allergic asthma. Attenuation ofeither pathway suppressed asthmatic symptoms. A better understanding ofthe BLT2-linked pathway and possible cross-regulation between the BLT1and BLT2 pathways should help to clarify their role in LTB₄-mediatedallergic pathogenesis. The finding that a LTB₄-BLT2-ROS pathway isinvolved in asthma serves as the basis for the development of newdiagnostic tools and treatments for allergic disease.

one aspect, the invention provides, a method of treating asthma in apatient, the method comprising administering to the patient atherapeutically effective amount of an agent that inhibits theexpression or intracellular signaling of long-form BLT2.

In another aspect, the invention provides a method of reducingexpression or activity of long-form BLT2 in a lung cell or immune cell,the method comprising contacting the lung cell or immune cell with aneffective amount of an agent that inhibits the expression orintracellular signaling of long-form BLT2.

In still another aspect, the invention provides a monoclonal antibodythat inhibits expression or intracellular signaling of long-form BLT2 ina lung cell or immune cell. In various embodiments, the monoclonalantibody is used for the treatment of asthma.

In a related aspect, the invention provides a pharmaceutical compositioncontains a monoclonal antibody that inhibits expression or intracellularsignaling of long-form BLT2 in a lung cell or immune cell and apharmaceutical carrier.

In yet another aspect, the invention provides a method for screening anagent for treating asthma, the method involving contacting a lung cellor immune cell containing a long-form BLT2 gene or long-form BLT2protein; and measuring the expression or activity of long-form BLT2,wherein a decrease in the expression or activity of long-form BLT2indicates the agent can be used for treating asthma.

In an additional aspect, the invention provides a kit for the treatmentof asthma, the kit comprising an agent that inhibits the expression orintracellular signaling of long-form BLT2.

In various embodiments of any of the aspects delineated herein, theagent selectively reduces the expression or intracellular signaling oflong-form BLT2 while the expression or intracellular signaling ofshort-form BLT2 is not disrupted. In various embodiments of any of theaspects delineated herein, the asthma is characterized by theoverexpression of long-form BLT2 in the lung airway. In variousembodiments of any of the aspects delineated herein, the expression oractivity of an asthma-associated Th2 cytokine selected from the groupconsisting of IL-13 is reduced. In various embodiments of any of theaspects delineated herein, one or more of eosinophil infiltration intolung airway, airway inflammation, or airway hyperresponsiveness (AHR) isreduced. In various embodiments of any of the aspects delineated herein,long-form BLT2 activation increases ROS generation and NF-KB activation.In various embodiments of any of the aspects delineated herein, themethod further comprises administering a therapeutically effectiveamount of an agent that inhibits the expression or activity of Rac.

In various embodiments of any of the aspects delineated herein, theagent is an antibody or fragment thereof that specifically bindslong-form BLT-2. In various embodiments, the antibody or fragmentthereof specifically binds long-form BLT-2 in the region set forth byamino acids 1-31 of SEQ ID NO: 3. In particular embodiments, theantibody or fragment thereof specifically binds long-form BLT-2 in theregion set forth by amino acids 14-27 of SEQ ID NO: 3. In variousembodiments of any of the aspects delineated herein, the antibody is apolyclonal or monoclonal antibody.

In various embodiments of any of the aspects delineated herein, theagent is an inhibitory nucleic acid that is complementary to at least aportion of a long-form BLT2 nucleic acid molecule. In variousembodiments, the inhibitory nucleic acid is complementary to at least aportion of a long-form BLT2 nucleic acid molecule in the region setforth by nucleotides 1-93 of SEQ ID NO: 2. In various embodiments of anyof the aspects delineated herein, the inhibitory nucleic acid isselected from the group consisting of an antisense molecule, and siRNA,and an shRNA.

Other objects and advantage of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the increased expression of BLT2 mRNA in an OVA-inducedmurine asthma model. FIG. 1A is a graph showing levels of LTB₄ in BALfluid measured using an ELISA at the indicated times following OVAchallenge. FIG. 1B shows semiquantitative RT-PCR analysis of BLT1 andBLT2. Levels of BLT2 mRNA in the lungs were measured in control(normal), pre-OVA challenged (pre-provocation) and OVA-challenged (OVAprovocation) mice. GAPDH was used as quantitative control. FIG. 1C showsimages of in situ hybridization of BLT2 mRNA in lung airways. Thedistributions of BLT2 mRNA in normal (panels i and ii) and OVA-inducedasthmatic mouse lung airways (panels iii and iv) are shown (arrows).Data are means±SEM (n=4 in each group). Magnification, 100 (panels i andii) or 200 (panels iii and iv).

FIGS. 2A-2D show that LY255283 attenuated airway inflammation in asthma.BALB/c mice were intravenously injected with LY255283 (2.5 mg/kg) orvehicle (DMSO) 1 hr before 1% OVA challenge. The mice were then killedon day 25 to assess asthmatic phenotypes. FIG. 2A shows semiquantitativeRT-PCR analysis of BLT2 mRNA levels in lung tissue.

FIG. 2B is a graph showing quantitative analyses of BLT2 mRNA levelsusing real-time PCR. FIG. 2C shows histological analysis of lung airwaysfrom OVA-challenged mice 48 hr after the last 1% OVA challenge. Lungswere excised, fixed, and stained with HE. Mice weresensitized/challenged with PBS (Normal, left panel), OVA with DMSOpretreatment (OVA/DMSO, middle panel), or OVA with LY255283 (2.5 mg/ml)pretreatment (OVA/LY255283, right panel). Scale Bars, 50 iAm. FIG. 2D isa graph showing histological scores. The data are means±SEM (n=5 in eachgroup). *P<0.05 vs. OVA/DMSO.

FIGS. 3A-3D show that antisense BLT2 attenuated airway inflammation inasthma.

FIG. 3A shows semiquantitative RT-PCR analysis of BLT2 mRNA levels inlung tissue. Normal and OVA-challenged mice (C57BL/6) pretreated withsense (ss) BLT2 (1.6 mg/kg), antisense (as) BLT2 (1.6 mg/kg) or buffer(saline) 24 hr and 1 hr before 10% OVA challenge were sacrificed twodays after the last OVA challenge, and their lungs were analyzed forlevels of BLT1, BLT2 and control GAPDH mRNAs using semiquantitativeRT-PCR analysis. FIG. 3B is a graph showing quantitative analyses ofBLT2 mRNA levels using real-time PCR. FIG. 3C are images showinginfiltration of eosinophils into BAL. Eosinophils (arrows) in BAL fluidwere obtained using cytospin and stained with Diff-Quick. Scale Bars, 50iAm. FIG. 3D depicts histological analysis of lung airways fromOVA-challenged mice 48 hr after OVA challenge.

Lungs were excised, fixed, and stained with HE. For this experiment,mice were pretreated with sense BLT2 (1.6 mg/kg; bottom left panel),antisense BLT2 (1.6 mg/kg; bottom right panel) or buffer (saline; topright panel) before OVA challenge. Normal lung tissue is shown at topleft panel. Scale Bars, 50 iAm. Data are means±SEM (n=5 in each group).*P<0.05 vs. OVA/ssBLT2; **P<0.01 vs. OVA/ssBLT2; ***P<0.001 vs.OVA/ssBLT2.

FIGS. 4A-4B show the effect of BLT2 inhibition on AHR. FIG. 4A shows theeffect of LY255283 on AHR in OVA-challenged mice. FIG. 4B shows theeffect of antisense BLT2 on AHR in OVA-challenged mice. AHR was measured24 h after the last 1% OVA challenge, after which mice was placed in achamber and nebulized with increasing doses of methacholine (6.25mg/ml-50 mg/ml) for 3 min. Data are means±SEM (n=5 in each group).*P<0.05 vs. OVA/DMSO in A or OVA/ssBLT2 in B.

FIGS. 5A-5B show that antisense BLT2 attenuated ROS generation. FIG. 5Adepicts FACS analysis of BLT2 expression in cells. BAL fluid wascollected 48 h after 10% OVA challenge. Normal and OVA-challenged micewere pretreated with sense BLT2 (1.6 mg/kg), antisense BLT2 (1.6 mg/kg)or buffer (saline) 24 hr and 1 hr before 10% provocation and thensacrificed at 48 hr after the last OVA challenge. The cells present inthe BAL fluid were washed and then immediately observed using aFACSCalibur™. FIG. 5B is a graph depicting measurement of LTB₄ levels inBAL fluid using a specific ELISA. Normal and OVA-challenged mice werepretreated with sense BLT2 (1.6 mg/kg), antisense BLT2 (1.6 mg/kg) orbuffer (saline) 24 h and 1 h before 10% provocation and then sacrificedat 48 h after the last OVA challenge. BAL fluid was then collected forLTB₄ analysis. Data are means±SEM (n=5 in each group). *P<0.05 vs.OVA/ssBLT2; **P<0.01 vs. OVA/ssBLT2.

FIGS. 6A-6E show that BLT2 inhibition attenuated NF-KB activation andVCAM-1 expression. FIG. 6A depicts EMSA analysis of NF-KB activationfollowing OVA-challenge.

Normal and OVA-challenged mice were pretreated with sense BLT2 (1.6mg/kg), antisense BLT2 (1.6 mg/kg) or buffer (saline) 24 hr and 1 hrbefore provocation and then sacrificed two days after the last OVAchallenge. Nuclear extracts were then prepared from the lungs andincubated with labeled NF-KB-binding oligonucleotides. FIG. 6B depictsWestern blot analysis of IKB-a expression in mouse model of asthmaadministered with asBLT2. FIG. 6C depicts Western blot analysis ofVCAM-1 in mouse model of asthma administered with asBLT2. Lung tissueextracts were prepared from normal mice, OVA-challenged mice(OVA/saline), OVA-challenged mice administered sense BLT2 (1.6 mg/kg) orantisense BLT2 (1.6 mg/kg). Equal amounts of protein were then analyzedby immunoblotting with antibodies against 1 KB-a (FIG. 6B) and VCAM-1(FIG. 6C). Tubulin was used as a loading control. FIG. 6D depictsWestern blot analysis of IKB-a expression in mouse model of asthmaadministered with LY255283. FIG. 6E depicts Western blot analysis ofVCAM-1 expression in mouse model of asthma administered with LY255283.Normal and OVA-challenged mice were pretreated with DMSO or LY255283(2.5 mg/kg) 1 hr before 1% OVA challenge and then sacrificed at 48 hrafter the last OVA challenge. Lung tissue extracts were then preparedfor Western blotting. Also shown the relative levels of 1 KB-a (FIG. 6D)and VCAM-1 (FIG. 6E) obtained using densitometry. The data are means±SEM(n=5 in each group).

FIG. 7 shows increased expression of BLT2 in bronchial biopsy specimens.Biopsy specimens were obtained from healthy subjects (left panels) andsubjects with mild (right panels) or moderate (bottom right panelbronchial asthma, after which the patterns of BLT2 expression insections of mucosa were visualized immunohistochemically. Positivesignals were colored red using a streptavidin-alkaline phosphatasesystem, and the cells were counter-stained using hematoxylin. The imagesshown are representative of experiments with similar results (n=4 forhealthy controls and mild bronchial asthma patients; n=5 for moderatebronchial asthma patients). Scale bars, 50 iAm. *P<0.05 vs. Normal.

FIGS. 8A and 8B show recruitment of T lymphocytes into airways. FIG. 8Adepicts FACS analysis showing recruitment of CD4+ T cells into the BALfluid of mice 12 hr after aerosol OVA challenge. FIG. 8 B depicts FACSanalysis showing recruitment of CD8+ T cells into the BAL fluid of mice12 hr after aerosol OVA challenge. BAL fluid was collected 12 hr after10% OVA challenge and washed with PBS. The leukocytes present werestained with FITC-conjugated anti-mouse TCR chain and PE-cy5 anti-mouseCD8a or PE rat anti-mouse CD4 after blocking with anti-FcRyantibody.Samples were then analyzed by flow cytometry to assess T lymphocyterecruitment. Data are means±SEM (n=6 in each group). *P<0.05 vs.OVA/asBLT2.

FIGS. 9A-9C show effect of antisense Rac oligonucleotide treatment onlung inflammation and NF-KB activation. FIG. 9A shows images of lungtissue in mouse model of asthma administered with asRac. To check theinvolvement of Rac in the process of eosinophil infiltration into thelung airway caused by OVA provocation, antisense oligonucleotides (1.25mg/kg of weight) were injected into the tail vein of the mice 24 hr and4 hr before provocation. Forty eight hours after OVA provocation, micewere sacrificed and BAL fluids and lung tissues were obtained. Cells inthe BAL fluid were attached to the slide glass and stained withHemacolour as manufacturer's recommendation. Lung tissues were obtainedfrom normal, buffer, control Rac oligonucleotide (control Rac) orantisense Rac oligonucleotide (asRac) treated mice after OVAprovocation. Lung tissues were fixed with 10% formaline, dehydrated andembedded in paraffin. The tissues were cut into 6 i.tm sections, andstained with Hematoxylin & Eosin. FIG. 9B depicts EMSA analysis of NF-KBin mouse model of asthma administered with asRac. To examine whether OVAprovocation induce the NF-KB activity and whether the induced NF-KB ismediated by Rac, electropholetic mobility shift assay (EMSA) wasaccompanied using antisense Rac oligonucleotide treated mice. Nuclearextracts were purified from the lung tissues and incubated with³²P-labeled double strand oligonucleotides containing NF-KB bindingconsensus or mutant sequence at room temperature. DNA-protein complexeswere separated by electrophoresis in 6% acrylamide gel undernondenaturing condition, the autoradiography was performed. FIG. 9Cdepicts Western blot analysis of VCAM-1 expression in mouse model ofasthma administered with asRac. To check expression level of VCAM-1,which is known to have critical role in the process of eosinophiltransendothelial migration from the blood vessel into the lungparenchyma whole cell lysates were obtained from the lung tissues andWestern blot analysis was carried out.

FIGS. 10A and 10B show activation of Rac in the lung tissue by OVAprovocation and inhibition of endogenous Rac expression by antisense Racoligonucleotide treatment. Lung samples were homogenized withmicropestle and washed with PBS twice. FIG. 10A depicts Western blotanalysis of Rac expression in mouse model of asthma. For membraneprotein preparation, cells were suspended in Buffer A and cells wereruptured by passing through 21-G syringe. After ultracentrifugation,protein samples in pelleted membrane fraction were dissolved with bufferA containing 1% Triton X-100. FIG. 10B depicts Western blot analysis ofRac expression in mouse model of asthma administered with asRac. Forwhole cell lysate preparation, cells from the lung tissues of buffer,control Rac oligonucleotide or antisense Rac oligonucleotide injectedmice were suspended with lysis buffer and incubated for 20 min. Aftercentrifugation of the samples, protein quantification was carried outusing Bradford reagent and Western blot analysis was performed.

FIG. 11A shows the suppression effect of BLT2 antisense oligonucleotideon BLT2 expression level by RT-PCR. FIG. 11B shows the suppressioneffect of BLT2 siRNA on BLT2 expression level by Northern blot.

FIGS. 12A and 12 b depict similar expression levels of LF-BLT2 orSF-BLT2 when transfected in CHO cells. FIG. 12A depicts RT-PCR analysisshowing that LF-BLT2 and SF-BLT2 transcript levels are similar. FIG. 12Bis a graph showing that LF-BLT2 or SF-BLT2 protein expression levelswere similar as determined by FACS analysis.

FIGS. 13A and 13B depict that LF-BLT2 is more active in mediatingchemotactic signaling and motility in CHO cells compared to SF-BLT2.FIG. 13A is a graph showing enhanced chemotactic motility of LF-BLT2transfected CHO cells in the presence of LTB4 compared to SF-BLT2transfected CHO cells. FIG. 13B is a graph showing enhanced ROSgeneration in LF-BLT2 transfected CHO cells in the presence of LTB₄compared to SF-BLT2 transfected CHO cells.

FIGS. 14A and 14B depict that growth and ERK activity were enhanced byLF-BLT2 compared to SF-BLT2. FIG. 14A depicts images of Rat-2 cellstransfected with empty vector (left panel), hLFBLT2 construct (middlepanel), and hSFBLT2 construct (right panel). FIG. 14B depicts Westernblot analysis of ERK in transfected cells, showing that Rat-2 cellstransfected with the hLFBLT2 construct had a significantly enhanced ERKactivation in the presence of LTB₄ compared to Rat-2 cells transfectedwith the hSF-BLT2 construct.

FIG. 15 depicts RT-PCR analysis of IL-13 (top panel) and GADPH (bottompanel) showing that LTB4-evoked IL-13 induction in bone marrow-derivedmast cells, indicating a role in allergic response.

FIGS. 16A and 16B depict the production of antibody to long form BLT2having BLT neutralizing activity. 253J-BV bladder cancer cells wereincubated with FITC-conjugated anti-BLT2 or an isotype control antibody,and BLT2 expression was evaluated by flow cytometry (red and greencolor). Fluorescence intensity of BLT2 expression level was measured.FIG. 16A depicts representative results of three independent experimentswith similar results. FIG. 16B is a chart showing that out of 22potential candidates, 6 BLT2 (long-form)-recognizing antibodies wereselected by FACS analysis. The 6 BLT2-recognizing antibodies aredesignated in red.

FIGS. 17A-17C are graphs depicting chemotaxis analysis using generatedanti-LF BLT2 antibody. FIG. 17A is a graph depicting the effect ofanti-LF BLT2 antibody on CHO or CHO-BLT2 stable cells exposed to 300 nMLTB₄ for 3 hr. LTB₄-induced chemotactic motility was determined in thepresence of BLT2 IgG Ab (BLT2-LF-26-22; 10 and 20 lug) and negativeantibody control (BLT2-LF-13 IgG Ab; 10 and 20 p.g). After migration,cells were fixed and stained with hematoxylin/eosin. FIG. 17B is a graphdepicting the effect of anti-LF BLT2 antibody on pcDNA3.1 or BLT2transfected CHO cells exposed to 300 nM LTB4 for 3 hr. LTB4-inducedchemotactic motility was determined in the presence of BLT2 IgG Ab(BLT2-LF-26-22; 10 and 20 lug) or control antibody (BLT2-LF-13 IgG Ab;10 and 20 p.g). After migration, cells were fixed and stained withhematoxylin/eosin. FIG. 17C is a graph showing that BLT1-inducedchemotactic migration was not affected by anti-BLT2 Ab (BLT2-LF-26-22),control Ab control (BLT2-LF-13, 20 lug), or pcDNA3.1. The BLT1transfected CHO cells were exposed to 10 nM LTB₄ for 3 hr with BLT2 IgGAb (BLT2-LF-26-22, 20 μg) and negative IgG Ab control (BLT2-LF-13, 20lig).

FIGS. 18A-18B depict AHT inhibition in asthma mouse model assay byanti-LF-BLT2 IgG antibody. FIG. 18A is a graph depicting the effect ofBLT2 inhibition on AHR. OVA-challenged mice were pretreated with controlantibody (100 μg/mice) and BLT2 IgG Ab (100 μg/mice, 500 μg/mice) 1 hrbefore 1% OVA challenge and then analyzed at 24 h after the last OVAchallenge. FIG. 18B is a graph showing that BLT2 IgG Ab attenuates ROSgeneration. BALF was collected 48 h after last OVA challenge.OVA-challenged mice were pretreated with control antibody (100 μg/mice),BLT2 IgG Ab (100 μg/mice, 500 μg/mice) 1 hr before 1% OVA challenge andthen sacrificed at 48 h after the last OVA challenge. The cells presentin the BALF were observed using a FACSCalibur.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally features compositions and methods fortreating asthma involving inhibitors of leukotriene B4 receptor BLT2,including long form BLT2.

BLT2 is a low-affinity receptor for leukotriene B4 (LTB4), a potentlipid mediator of inflammation generated from arachidonic acid via the5-lipoxygenase pathway. Unlike BLT1, a high-affinity receptor for LTB4,no physiological role has yet been identified for BLT2, especially withregard to the pathogenesis of asthma. A murine model of allergic asthmawas used to evaluate the role of BLT2 in ovalbumin-induced airwayinflammation and airway hyperresponsiveness (AHR). The levels of BLT2mRNA and its ligand LTB₄ in the lung airway were highly elevated afterOVA challenge, and downregulation of BLT2 with antisense BLT2oligonucleotides markedly attenuated the airway inflammation and AHR,suggesting a role of BLT2 in the asthmatic response. Further analysisaimed at identifying mediators downstream of BLT2 revealed that BLT2activation led to elevation of reactive oxygen species (ROS) andsubsequent activation of NF-KB, thus inducing the expression of VCAM-1that is known to be involved in eosinophil infiltration into lungairway. Together the findings suggest that BLT2 plays a pivotal role inthe pathogenesis of asthma, acting through a ‘ROS-NF-KB’-linkedsignaling pathway. Finally, immunohistochemical assay of clinicalsubjects demonstrated that BLT2 expression was high in the airwayepithelial layers as well as the microvascular endothelium, as in themurine model of asthma.

According to one aspect of the present invention, there is provided ause of a substance that inhibits the expression or intracellularsignaling of BLT2 for the manufacture of a medicament for the treatmentof asthma. In this specification, the phrase “inhibit(s) the expressionof BLT2” means to inhibit any step among the transcription, mRNAprocessing, translation, translocation, and maturation of BLT2, and thephrase “inhibit(s) the intracellular signaling of BLT2” means to inhibitany step among the binding of LTB₄ to BLT2, the activation of BLT2 andits intracellular signaling pathway to induce asthma.

The nucleotide sequence of human BLT2 gene is available at the NCBI(NM_019839) and denoted as SEQ ID NO: 1 in this specification. The BLT2gene has 2 kinds of CDS form, long form CDS (1618-2787) and short formCDS (1711-2787), the nucleotide sequences of which are denoted as SEQ IDNO: 2 and SEQ ID NO: 4, respectively. The amino acid sequence of thelong form BLT2 protein is available at the NCBI (NM_019839) and isdenoted as SEQ ID NO: 3. The amino acid sequence of the long form BLT2protein is available at the NCBI (AB029892) and is denoted as SEQ ID NO:5.

In a preferred embodiment, the substance may be an antibody to BLT2(e.g., long form BLT2). The antibody to BLT2 inhibits the intracellularsignaling of BLT2. The antibody binds to BLT2 competitively with LTB₄,so that can inhibit the intracellular signaling of BLT2. The antibodycan be produced according to the conventional methods for producingpolyclonal or monoclonal antibody by using BLT2 or its fragment as anantigen.

In a preferred embodiment, the substance may be a compound that binds toBLT2 and inhibits the intracellular signaling of BLT2. The compound isalso referred to as BLT2 antagonist, which means a compound thatantagonizes an action of LTB₄ on BLT2. The compound can be screenedaccording to the present screening method from the commerciallyavailable chemical DB.

In a preferred embodiment, the compound may be LY255283(145-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxylphenyll-ethanone).LY255283 is a competitive antagonist of the BLT2 receptor. LY255283 havebeen known to inhibit eosinophil chemotaxis by 80% at a concentration of10 1 AM, and inhibits the binding of radiolabeled LTB₄ to eosinophilmembranes with an IC50 of 260 nM [Ann N Y Acad Sci 629 274-287 (1991)].Also, LY255283 have been known to be a novel leukotriene B4 receptorantagonist, which limits activation of neutrophils and prevents acutelung injury induced by endotoxin in pigs [Surgery. 1993 August;114(2):191-8]. However, the anti-asthma activity of LY25583 was revealedby the present inventors for the first time.

In a preferred embodiment, the substance may be an antisense or siRNAoligonucleotide that inhibits the expression of BLT2. The antisense orsiRNA oligonucleotide has a nucleotide sequence complementary to thenucleotide sequence of BLT2 mRNA as set forth in SEQ ID NO: 2.

The term “antisense oligonucleotide” used herein is intended to refer tonucleic acids, preferably, DNA, RNA or its derivatives, that arecomplementary to the nucleotide sequences of a target mRNA,characterized in that they binds to the target mRNA and interfere itstranslation to protein. The antisense oligonucleotide of this inventionmeans DNA or RNA sequences complementary and binding to BLT2 mRNA, thatare able to inhibit translation, translocation, maturation or otherbiological functions of BLT2 mRNA. The antisense nucleic acid is 6-100,preferably, 8-60, more preferably, 10-40 nucleotides in length.

The antisense oligonucleotide may comprise at lease one modification inits base, sugar or backbone for its higher inhibition efficacy (DeMesmaeker et al., Curr Opin Struct Biol., 5(3):343-55(1995)). Themodified nucleic acid backbone comprises phosphorothioate,phosphotriester, methyl phosphonate, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. The antisense oligonucleotide may also contain oneor more substituted sugar moieties. The antisense nucleic acid mayinclude one or more modified bases, for example, hypoxanthine,6-methyladenine, 5-me pyrimidines (particularly, 5-methylcytosine),5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as wellas synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N⁶(6-aminohexyl)adenine and 2,6-diaminopurine. Another modification ofthe oligonucleotides of the invention involves chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 86:6553(1989)), cholic acid (Manoharan et al. Bioorg. Med. Chem.Let, 4:1053(1994)), a thioether, e.g., hexyl-S-tritylthiol (Manoharan etal. Ann. NY. Acad. ScL, 660:306(1992); Manoharan et al. Bioorg. Med.Chem. Let, 3: 2765(1993)), a thiocholesterol (Oberhauser et al., Nucl.Acids Res., 20:533(1992)), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al. EMBO J., 10:111(1991); Kabanovet al. FEBS Lett, 259:327(1990); Svinarchuk et al. Biochimie,75:49(1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al. Tetrahedron Lett, 36:3651(1995); Shea et al. Nucl.Acids Res., 18:3777(1990)), a polyamine or a polyethylene glycol chain(Manoharan et al. Nucleosides & Nucleotides, 14:969(1995)), oradamantane acetic acid (Manoharan et al. Tetrahedron Lett., 36:3651(1995)). Oligonucleotides comprising lipophilic moieties, andmethods for preparing such oligonucleotides are known in the art, forexample, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255. Themodifications described above enhance stability against nucleasedegradation and increase affinity of the antisense oligonucleotidetoward its target mRNA.

The antisense molecule is conventionally synthesized in vitro and thentransmitted to cells. In addition, it is intracellular^ produced bytranscription from foreign sequence. In vitro synthesis involves RNApolymerase I. In vivo transcription for preparing antisense RNA usesvector having origin of recognition region (MCS) in oppositeorientation. The antisense RNA preferably comprises a translation stopcodon for inhibiting translation to peptide.

According to a preferred embodiment, the antisense oligonucleotide mayhave a nucleotide sequence of SEQ ID NO: 6, which is complementary tothe target region (1738-1752) of SEQ ID NO: 2.

According to a preferred embodiment, the siRNA oligonucleotide may havea sense sequence of SEQ ID NO: 7 and an antisense sequence of SEQ ID NO:8, which is complementary to the target region (1705-1724) of SEQ ID NO:2.

The term “siRNA” used herein refers to a nucleic acid molecule mediatingRNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). The siRNA toinhibit expression of a target gene provides effective gene knock-downmethod or gene therapy method. It was been first in plants, insects,Drosophila melanogaster and parasites and recently has been used formammalian cell researches. The siRNA molecule of this invention mayconsist of a sense RNA strand (having sequence corresponding to BLT2mRNA) and an antisense RNA strand (having sequence complementary to BLT2mRNA) and form a duplex structure.

Alternatively, the siRNA molecule of this invention may have a singlestrand structure comprising self-complementary sense and antisensestrands.

The siRNA of this invention is not restricted to a RNA duplex of whichtwo strands are completely paired and may comprise non-paired portionsuch as mismatched portion with non-complementary bases and bulge withno opposite bases. The overall length of the siRNA is 10-100nucleotides, preferably, 15-80 nucleotides, and more preferably, 20-70nucleotides.

The siRNA may comprise either blunt or cohesive end so long as itenables to silent the BLT2 expression due to RNAi effect. The cohesiveend may be prepared in 3′-end overhanging structure or 5′-endoverhanging structure.

The siRNA may be constructed by inserting a short nucleotide sequence(e.g., about 5-15 nt) between self-complementary sense and antisensestrands. The siRNA expressed forms a hairpin structure by intramolecularhybridization, resulting in the formation of stem-and-loop structure.The stem-and-loop structure is processed in vitro or in vivo to generateactive siRNA molecule mediating RNAi.

In a preferred embodiment, the substance may be a compound that inhibitsthe upstream or downstream signaling pathway of BLT2. In certainembodiments, a compound of the invention can prevent, inhibit, ordisrupt, or reduce by at least 10%, 25%, 50%, 75%, or 100% the activityof a BLT2 pathway by binding to BLT, e.g., long-form BLT2. Ananti-asthma therapeutic, such as antibody against long-form BLT2, may beadministered in combination with any other standard therapy orconventional agent for treating asthma; such methods are known to theskilled artisan and described in Remington's Pharmaceutical Sciences byE. W. Martin

In the preferred embodiment, the asthma may be characterized by thatBLT2 protein is over-expressed in the lung airway. The present inventorshave found that BLT2 protein and its ligand LTB4 were over-expressed inthe lung airway after OVA challenge, and downregulation of BLT2 withantisense BLT2 oligonucleotides markedly attenuated the airwayinflammation and AHR. Therefore, any anti-asthma therapy strategy basedon the inhibition of BLT2 overexpression is claimed as the presentinvention.

In the preferred embodiment, the over-expression, i.e. activation ofBLT2 may cause asthmatic symptoms by elevating ROS generation andsubsequent NF-KB activation. The present inventors demonstrated that theBLT2 activation led to elevation of reactive oxygen species (ROS) andsubsequent activation of NF-KB, thus inducing the expression of VCAM-1that is known to be involved in eosinophil infiltration into lungairway.

In the preferred embodiment, the treatment of asthma may be accomplishedby reducing eosinophil infiltration into lung airway, airwayinflammation and airway hyperresponsiveness (AHR). Therefore, any use ofBLT2 inhibitors as a therapeutic composition against asthma is claimedin the present invention.

According to another aspect of the present invention, there is provideda use of a combination of (a) a substance that inhibits the expressionor intracellular signaling of BLT2, and (b) other anti-asthma drugs forthe manufacture of a medicament for the treatment of asthma. Accordingto another aspect of the present invention, there is provided a use of asubstance that inhibits the expression or activity of Rac for themanufacture of a medicament for the treatment of asthma. In thisspecification, the phrase “inhibit(s) the expression of Rac” means toinhibit any step among the transcription, mRNA processing, translation,translocation, and maturation of Rac, and the phrase “inhibit(s) theactivity of Rac” means to inhibit any step among the GTPase activity ofRac and its intracellular signaling pathway to induce asthma.

Rac, a member of Rho family GTPases, mediates various cellular responsessuch as actin polymerization, cell proliferation, cPLA2 activation, andgeneration of reactive oxygen species (ROS). A mouse model system forasthma was used to determine the role of Racl on allergen-inducedbronchial inflammation and airway hyperresponsiveness (AHR). Raclactivity is dramatically stimulated after allergen challenge andadministration of antisense oligomers to Racl remarkably reducedbronchial inflammation and AHR. In a further study to determine thesignaling mechanism by which Racl mediates asthmatic inflammation andAHR, Racl was important for the NFkB activation critically implicated inthe transcription of various inflammatory genes such as VCAM-1.Additionally, Racl was shown to mediate the activation of cPLA2, whichcatalyzes the hydrolysis of membrane phospholipids leading to therelease of arachidonic acid (AA) and subsequently eicosanoids such asleukotrienes (LTs). Together, these findings indicate that Rad iscritically involved in the pathogenesis of the bronchial asthma. In thepreferred embodiment, the substance may be an antisense or siRNAoligonucleotide that inhibits the expression of Rac. The antisense orsiRNA oligonucleotide has a nucleotide sequence complementary to thenucleotide sequence of Rac mRNA as set forth in SEQ ID NO: 13. Thesequence of mRNA or CDS of human Rac gene is available at the NCBI(gi:156071511) and its deduced amino acid sequence is denoted as SEQ IDNO: 14.

According to another aspect of the present invention, there is provideda pharmaceutical composition for the treatment of asthma, whichcomprises a substance that inhibits the expression or intracellularsignaling of BLT2 as an active ingredient. In the pharmaceuticalcomposition of the present invention, the substance may be chemicalcompounds, peptides, antibody proteins, nucleotides, antisenseoligonucleotides, siRNA oligonucleotides or extract of natural source.The present pharmaceutical composition may comprise a pharmaceuticallyacceptable carrier in addition.

According to another aspect of the present invention, there is provideda pharmaceutical composition for the treatment of asthma, whichcomprises a substance that inhibits the expression or activity of Rac asan active ingredient. In the pharmaceutical composition of the presentinvention, the substance may be chemical compounds, peptides, antibodyproteins, nucleotides, antisense oligonucleotides, si RNAoligonucleotides or extract of natural source. The presentpharmaceutical composition may comprise a pharmaceutically acceptablecarrier in addition.

According to another aspect of the present invention, there is provideda method for treating a patient with asthma, which comprisesadministering a therapeutically effective amount of a substance thatinhibits the expression or intracellular signaling of BLT2 to thepatient.

According to another aspect of the present invention, there is provideda method for treating a patient with asthma, which comprisesadministering a therapeutically effective amount of a substance thatinhibits the expression or activity of Rac to the patient.

According to another aspect of the present invention, there is provideda method for screening a substance for treating asthma, which comprisesthe steps of: (a) contacting the substance to be analyzed to a cellcontaining BLT2 gene or protein; and, (b) measuring the expression orintracellular signaling level of BLT2, wherein if the expression orintracellular signaling level of BLT2 is down-regulated, the substanceis determined to have a potency to treat asthma.

According to the present method, the cell containing the BLT2 gene orprotein can be easily prepared by obtaining cells containing theiroriginal BLT2 gene or by transfecting cells with a foreign BLT 2 gene.The cells containing the BLT2 gene or protein are first contacted tosubstances to be analyzed. The term “substance” used herein inconjunction with the present screening method refers to a materialtested in the present method for analyzing the influence on theexpression level of the BLT2 gene, the amount of the BLT2 protein or theintracellular signaling level of the BLT2 receptor. The substanceincludes chemical compounds, peptides, antibody proteins, nucleotides,antisense-RNA, siRNA (small interference RNA) and extract of naturalsource, but not limited to.

Afterwards, the expression level of the BLT2 gene, the amount of theBLT2 protein or the intracellular signaling level of the BLT2 receptorin cells is measured. Where the expression level of the BLT2 gene, theamount of the BLT2 protein or the intracellular signaling level of theBLT2 receptor is measured to be down-regulated, the substance isdetermined to be a candidate to treat asthma.

The measurement of the expression level of the BLT2 gene could becarried out by a variety of methods known in the art. For example,RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press(2001)), Northern blotting (Peter B. Kaufma etal., Molecular and Cellular Methods in Biology and Medicine, 102-108,CRC press), hybridization using cDNA microarray (Sambrook et al.,Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring HarborPress(2001)) and in situ hybridization (Sambrook et al., MolecularCloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press(2001))may be used. Where the expression level of the BLT2 gene is analyzed byRT-PCT, total RNA is first isolated from cells treated with a substanceto be analyzed and a first cDNA strand is then synthesized using oligodT primer and reverse transcriptase. Then, PCR amplifications areperformed using the first cDNA strand as templates and a BI_T2-specificprimer set. Finally, the PCR amplified products are resolved byelectrophoresis and bands are analyzed for assessing the expressionlevel of the BLT2 gene.

The amount of the BLT2 protein may be determined by various immunoassaysknown in the art. For example, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),capture-ELISA, inhibition or competition assay and sandwich assay areused for analyzing the amount of the BLT2 protein.

The intracellular signaling level of the BLT2 receptor may be determinedby monitoring an event induced by LTB4, e.g., monitoring the rise of theintracellular calcium concentration as described in example usingBLT2-expressing cells etc. (e.g., BLT2 overexpressing cells etc.). Forexample, if the substance reduces the intracellular calciumconcentration by LTB4 in BLT2-expressing cells, it can be judged as BLT2antagonist.

According to another aspect of the present invention, there is provideda method for screening a substance for treating asthma, which comprisesthe steps of: (a) contacting the substance to be analyzed to a cellcontaining Rac gene or protein; and, (b) measuring the expression oractivity level of Rac, wherein if the expression or activity level ofRac is down-regulated, the substance is determined to have a potency totreat asthma.

According to another aspect of the present invention, there is provideda kit for detecting asthma, which comprises a primer or probe having anucleotide sequence complementary to the nucleotide sequence of BLT2 asset forth in SEQ ID NO: 2. Therefore, any methodology or kit developedbased on the information that BLT2 overexpression is detected in thelung airway of a patient with asthma may be included in the presentinvention.

The probes or primers used in the present kit has a complementarysequence to the nucleotide sequence of the BLT2 gene. The term“complementary” with reference to sequence used herein refers to asequence having complementarity to the extent that the sequence annealsor hybridizes specifically with the nucleotide sequence of the BLT2 geneunder certain annealing or hybridization conditions. In this regard, theterm “complementary” used herein has different meaning from the term“perfectly complementary”. The probes or primers used in the presentinvention can be one or more mismatch, so long as such mismatches arenot sufficient to completely preclude specific annealing orhybridization to the BLT2 gene.

As used herein the term “probe” means a linear oligomer of natural ormodified monomers or linkages, including deoxyribonucleotides andribonucleotides, capable of specifically binding to a targetpolynucleotide. The probe may be naturally occurring or artificiallysynthesized. The probe is preferably single stranded. Preferably, theprobes used in the present invention are oligodeoxyribonucleotides. Theprobe of this invention can be comprised of naturally occurring dNMP(i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide, or non-naturalnucleotide. The primer can also include ribonucleotides. For instance,the probes of this invention may include nucleotides with backbonemodifications such as peptide nucleic acid (PNA) (M. Egholm et al.,Nature, 365:566-568(1993)), phosphorothioate DNA, phosphorodithioateDNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2-0-methylRNA, alpha-DNA and methylphosphonate DNA, nucleotides with sugarmodifications such as 2′-0-methyl RNA, 2′-fluoro RNA, 2′-amino RNA,2′-0-alkyl DNA, 2′40-allyl DNA, 2′-0-alkynyl DNA, hexose DNA, pyranosylRNA, and anhydrohexitol DNA, and nucleotides having base modificationssuch as C-5 substituted pyrimidines (substituents including fluoro-,bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-,propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), 7-deazapurineswith C-7 substituents (substituents including fluoro-, bromo-, chloro-,iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-,imidazolyl-, pyridyl-), inosine, and diaminopurine.

The term “primer” as used herein refers to an oligonucleotide, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of primer extension product which iscomplementary to a nucleic acid strand (template) is induced, i.e., inthe presence of nucleotides and an agent for polymerization, such as DNApolymerase, and at a suitable temperature and pH. The suitable length ofprimers will depend on many factors, including temperature, applicationand source of primer, generally, 15-30 nucleotides in length. Shorterprimers generally need lower temperature to form stable hybridizationduplexes to templates.

The sequences of primers are not required to have perfectlycomplementary sequence to templates. The sequences of primers maycomprise some mismatches, so long as they can be hybridized withtemplates and serve as primers. Therefore, the primers of this inventionare not required to have perfectly complementary sequence to the BLT2gene as templates; it is sufficient that they have complementarity tothe extent that they anneals specifically to the nucleotide sequence ofthe BLT2 gene for acting as a point of initiation of synthesis. Theprimer design may be conveniently performed with referring to the BLT2gDNA or cDNA sequences, preferably, cDNA sequence. For instance, theprimer design may be carried out using computer programs for primerdesign (e.g., PRIMER 3 program). Exemplified primers of this inventionis set forth in SEQ ID NO: 9 (sense primer) and SEQ ID NO: 10 (antisenseprimer).

According to a preferred embodiment, the diagnosis or detection kit forasthma comprising probes is in the form of microarray, more preferablyDNA or cDNA microarray, most preferably cDNA microarray.

In microarray, the present probes serve as hybridizable array elementsand are immobilized on substrates. A preferable substrate includessuitable solid or semisolid supporters, such as membrane, filter, chip,slide, wafer, fiber, magnetic or nonmagnetic bead, gel, tubing, plate,macromolecule, microparticle and capillary tube. The hybridizable arrayelements are arranged and immobilized on the substrate. Suchimmobilization occurs through chemical binding or covalent binding suchas UV. In an embodiment of this invention, the hybridizable arrayelements are bound to a glass surface modified to contain epoxi compoundor aldehyde group or to a polylysin-coated surface. Further, thehybridizable array elements are bound to a substrate through linkers(e.g. ethylene glycol oligomer and diamine).

DNAs to be examined with a microarry of this invention may be labeled,and hybridized with array elements on microarray. Various hybridizationconditions are applicable, and for the detection and analysis of theextent of hybridization, various methods are available depending onlabels used.

The present method for diagnosing rheumatoid arthritis may be carriedout in accordance with hybridization. For such analysis, probes, whichhave a complementary sequence to the nucleotide sequence of the BLT2gene, are used.

Using probes hybridizable with the BLT2 gene or cDNA, preferably cDNA,asthma is diagnosed or detected by hybridization-based assay. Accordingto a preferred embodiment, some modifications in the probes of thisinvention can be made unless the modifications abolish the advantages ofthe probes. Such modifications, i.e., labels linking to the probesgenerate a signal to detect hybridization. Suitable labels includefluorophores (e.g., fluorescein), phycoerythrin, rhodamine, lissamine,Cy3 and Cy5 (Pharmacia), chromophores, chemiluminescers, magneticparticles, radioisotopes (e.g., P³² and S³⁵), mass labels, electrondense particles, enzymes (e.g., alkaline phosphatase and horseradishperoxidase), cofactors, substrates for enzymes, heavy metals (e.g.,gold), and haptens having specific binding partners, e.g., an antibody,streptavidin, biotin, digoxigenin and chelating group, but not limitedto. Labeling is performed according to various methods known in the art,such as nick translation, random priming (Multiprime DNA labelingsystems booklet, “Amersham” (1989)) and kination (Maxam & Gilbert,Methods in Enzymology, 65:499(1986)). The labels generate signaldetectable by fluorescence, radioactivity, measurement of colordevelopment, mass measurement, X-ray diffraction or absorption, magneticforce, enzymatic activity, mass analysis, binding affinity, highfrequency hybridization or nanocrystal.

The nucleic acid sample (preferably, cDNA) to be analyzed may beprepared using mRNAfrom various biosamples. The biosample is preferablya cell from airway epithelium. Instead of probes, cDNA may be labeledfor hyribridization-based analysis.

Probes are hybridized with cDNA molecules under stringent conditions fordetecting asthma. Suitable hybridization conditions may be routinelydetermined by optimization procedures. Conditions such as temperature,concentration of components, hybridization and washing times, buffercomponents, and their pH and ionic strength may be varied depending onvarious factors, including the length and GC content of probes andtarget nucleotide sequence.

The detailed conditions for hybridization can be found in JosephSambrook, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M. L. M.Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999). For example, the high stringent condition includes hybridizationin 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA at 65° C.and washing in 0.1×SSC (standard saline citrate)/0.1% SDS at 68° C.Also, the high stringent condition includes washing in 6×SSC/0.05%sodium pyrophosphate at 48° C. The low stringent condition includese.g., washing in 0.2×SSC/0.1% SDS at 42° C.

Following hybridization reactions, a hybridization signal indicative ofthe occurrence of hybridization is then measured. The hybridizationsignal may be analyzed by a variety of methods depending on labels. Forexample, where probes are labeled with enzymes, the occurrence ofhybridization may be detected by reacting substrates for enzymes withhybridization resultants. The enzyme/substrate pair useful in thisinvention includes, but not limited to, a pair of peroxidase (e.g.,horseradish peroxidase) and chloronaphtol, aminoethylcarbazol,diaminobenzidine, D-luciferin, lucigenin (bis-A/-methylacridiniumnitrate), resorufin benzyl ether, luminol, Amplex Red reagent(10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl andpyrocatechol), TMB (3,3,5,5-tetramethylbenzidine), ABTS(2,2-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine(OPD) or naphtol/pyronine; a pair of alkaline phosphatase andbromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT),naphthol-AS-B1-phosphate or ECF substrate; and a pair of glucosidase andt-NBT (nitroblue tetrazolium) or m-PMS (phenzaine methosulfate). Whereprobes are labeled with gold particles, the occurrence of hybridizationmay be detected by silver staining method using silver nitrate.

In these connections, where the present method for diagnosing asthma iscarried out by hybridization, it comprises the steps of (i) contacting anucleic acid sample to a probe having a nucleotide sequencecomplementary to the nucleotide sequence of the BLT2 gene; and (ii)detecting the occurrence of hybridization.

The signal intensity from hybridization is indicative of asthma. Whenthe hybridization signal to BLT2 cDNA from a sample to be diagnosed ismeasured to be stronger than normal samples, the sample can bedetermined to have asthma.

According to a preferred embodiment, the primers of this invention areused for amplification reactions.

The term used herein “amplification reactions” refers to reactions foramplifying nucleic acid molecules. A multitude of amplificationreactions have been suggested in the art, including polymerase chainreaction (hereinafter referred to as PCR) (U.S. Pat. Nos. 4,683,195,4,683,202, and 4,800,159), reverse transcription-polymerase chainreaction (hereinafter referred to as RT-PCR) (Sambrook, J. et al.,Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring HarborPress(2001)), the methods of Miller, H. I. (WO 89/06700) and Davey, C.et al. (EP 329,822), ligase chain reaction (LCR)(17, 18), Gap-LCR (WO90/01069), repair chain reaction (EP 439,182), transcription-mediatedamplification (TMA)(19) (WO 88/10315), self sustained sequencereplication (WO 90/06995), selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos.5,413,909 and 5,861,245), nucleic acid sequence based amplification(NASBA) (U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603),strand displacement amplification and loop-mediated isothermalamplification (LAMP), but not limited to. Other amplification methodsthat may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,4,988,617 and in U.S. Ser. No. 09/854,317.

According to the most preferred embodiment, the amplification reactionis carried out in accordance with PCR (polymerase chain reaction) whichis disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.

PCR is one of the most predominant processes for nucleic acidamplification and a number of its variations and applications have beendeveloped. For example, for improving PCR specificity or sensitivity,touchdown PCR(24), hot start PCR(25, 26), nested PCR(2) and boosterPCR(27) have been developed with modifying traditional PCR procedures.In addition, real-time PCR, differential display PCR (DD-PCR), rapidamplification of cDNA ends (RACE), multiplex PCR, inverse polymerasechain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR(TAIL-PCR) and multiplex PCR have been suggested for certainapplications. The details of PCR can be found in McPherson, M J., andMoller, S. G. PCR. BIOS Scientific Publishers, Springer-Verlag New YorkBerlin Heidelberg, N.Y. (2000), the teachings of which are incorporatedherein by reference in its entity.

Where the present method for diagnosing asthma is carried out usingprimers, the nucleic acid amplification is executed for analyzing theexpression level of the BLT2 gene. Because the present invention isintended to assess the expression level of the BLT2 gene, the level ofthe BLT2 mRNA in samples is analyzed.

Therefore, the present invention performs nucleic acid amplificationsusing mRNA molecules in samples as templates and primers to be annealedto mRNA or cDNA.

For obtaining mRNA molecules, total RNA is isolated from samples. Theisolation of total RNA may be performed by various methods (Sambrook, J.et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold SpringHarbor Press(2001); Tesniere, C. et al., Plant Mol. Biol. Rep.,9:242(1991); Ausubel, F. M. et al., Current Protocols in MolecularBiology, John Willey & Sons(1987); and Chomczynski, P. et al., Anal.Biochem. 162:156(1987)). For example, total RNA in cells may be isolatedusing Trizol. Afterwards, cDNA molecules are synthesized using mRNAmolecules isolated and then amplified. Since total RNA molecules used inthe present invention are isolated from human samples, mRNA moleculeshave poly-A tails and converted to cDNA by use of dT primer and reversetranscriptase (PNAS USA, 85:8998(1988); Libert F, et al., Science,244:569(1989); and Sambrook, J. et al., Molecular Cloning. A LaboratoryManual, 3rd ed. Cold Spring Harbor Press(2001)). cDNA moleculessynthesized are then amplified by amplification reactions.

The primers used for the present invention is hybridized or annealed toa region on template so that double-stranded structure is formed.Conditions of nucleic acid hybridization suitable for forming suchdouble stranded structures are described by Joseph Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985).

A variety of DNA polymerases can be used in the amplification step ofthe present methods, which includes “Klenow” fragment of E. coli DNApolymerase I, a thermostable DNA polymerase and bacteriophage T7 DNApolymerase. Preferably, the polymerase is a thermostable DNA polymerasesuch as may be obtained from a variety of bacterial species, includingThermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis,Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).

When a polymerization reaction is being conducted, it is preferable toprovide the components required for such reaction in excess in thereaction vessel. Excess in reference to components of the amplificationreaction refers to an amount of each component such that the ability toachieve the desired amplification is not substantially limited by theconcentration of that component. It is desirable to provide to thereaction mixture an amount of required cofactors such as Mg2±, and dATP,dCTP, dGTP and dTTP in sufficient quantity to support the degree ofamplification desired. All of the enzymes used in this amplificationreaction may be active under the same reaction conditions. Indeed,buffers exist in which all enzymes are near their optimal reactionconditions. Therefore, the amplification process of the presentinvention can be done in a single reaction volume without any change ofconditions such as addition of reactants.

Annealing or hybridization in the present method is performed understringent conditions that allow for specific binding between the primerand the template nucleic acid. Such stringent conditions for annealingwill be sequence-dependent and varied depending on environmentalparameters.

The amplified BLT2 cDNA molecules are then analyzed to assess theexpression level of the BLT2 gene. For example, the amplified productsare resolved by a gel electrophoresis and the bands generated areanalyzed to assess the expression level of the BLT2 gene. When theexpression level of the BLT2 gene from a sample to be diagnosed ismeasured to be higher than normal samples, the sample can be determinedto have asthma.

In these connections, where the present method for diagnosing asthma iscarried out by amplification, it comprises the steps of (i) amplifying anucleic acid sample by use of a primer having a nucleotide sequencecomplementary to the nucleotide sequence of the BLT2 gene; and (ii)analyzing the amplified products to determine the expression level ofthe BLT2 gene.

In a preferred embodiment, the kit may comprise a pair of primers havinga forward sequence of SEQ ID NO: 9 and a reverse sequence of SEQ ID NO:10. This primer set can detect both of the long form and short formBLT2.

In a preferred embodiment, the kit may comprise a pair of primers havinga forward sequence of SEQ ID NO: 11 and a reverse sequence of SEQ ID NO:12. This primer set can detect only long form of BLT2 because the primerrecognizes the front part of long form CDS. According to another aspectof the present invention, there is provided a kit for detecting asthma,which comprises an antibody binding specifically to BLT2 protein. Thediagnosing kit for asthma may be constructed by incorporating anantibody binding specifically to the BLT2 protein.

The antibody against the BLT2 protein used in this invention maypolyclonal or monoclonal, preferably monoclonal. The antibody could beprepared according to conventional techniques such as a fusion method(Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)),a recombinant DNA method (U.S. Pat. No. 4,816,56) or a phage antibodylibrary (Clackson et al, Nature, 352:624-628(1991) and Marks et al, J.Mol. Biol., 222:58, 1-597(1991)).

The general procedures for antibody production are described in Harlow,E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring HarborPress, New York, 1988; Zola, H., Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc., Boca Raton, Fla., 1984; and Coligan,CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N Y, 1991, which areincorporated herein by references. For example, the preparation ofhybridoma cell lines for monoclonal antibody production is done byfusion of an immortal cell line and the antibody producing lymphocytes.This can be done by techniques well known in the art. Polyclonalantibodies may be prepared by injection of the BLT2 protein antigen tosuitable animal, collecting antiserum containing antibodies from theanimal, and isolating specific antibodies by any of the known affinitytechniques.

Where the diagnosing method of this invention is performed usingantibodies to the BLT2 protein, it could be carried out according toconventional immunoassay procedures for detecting asthma.

Such immunoassay may be executed by quantitative or qualitativeimmunoassay protocols, including radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),capture-ELISA, inhibition or competition assay, sandwich assay, flowcytometry, immunofluorescence assay and immuoaffinity assay, but notlimited to. The immunoassay and immuostaining procedures can be found inEnzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla.,1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methodsin Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, N J,1984; and Ed Harlow and David Lane, Using Antibodies: A LaboratoryManual, Cold Spring Harbor Press, 1999, which are incorporated herein byreferences.

For example, according to the radioimmunoassay method, the radioisotope(e.g., c^(14, 1125, P32) and S³⁵) labeled antibody may be used to detectthe BLT2 protein.

In addition, according to the ELISA method, the example of the presentmethod may comprise the steps of: (i) coating a surface of solidsubstrates with cell lysate to be analyzed; (ii) incubating the coatedcell lysate with a primary antibody to the BLT2 protein; (iii)incubating the resultant with a secondary antibody conjugated with anenzyme; and (iv) measuring the activity of the enzyme.

The solid substrate useful in this invention includes carbohydratepolymer (e.g., polystyrene and polypropylene), glass, metal and gel,most preferably microtiter plates.

The enzyme conjugated with the secondary antibody is that catalyzingcolorimetric, fluorometric, luminescence or infra-red reactions, e.g.,including alkaline phosphatase, f3-galactosidase, luciferase, CytochromeP450 and horseradish peroxidase. Where using alkaline phosphatase,bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT) or ECFmay be used as a substrate for color-developing reactions; in the caseof using horseradish peroxidase, chloronaphtol, aminoethylcarbazol,diaminobenzidine, D-luciferin, lucigenin (bis-W-methylacridiniumnitrate), resorufin benzyl ether, luminol, Amplex Red reagent(10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl andpyrocatechol), TMB (3,3,5,5-tetramethylbenzidine), ABTS(2,2-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine(OPD) or naphtol/pyronine may be used as a substrate; and in the case ofusing glucose oxidase, t-NBT (nitroblue tetrazolium) or m-PMS (phenzainemethosulfate) may be used as a substrate.

Where the present method is performed in accordance with thecapture-ELISA method, the specific example of the present method maycomprise the steps of: (i) coating a surface of a solid substrate with acapturing antibody capable of binding specifically to the BLT2 protein;(ii) incubating the capturing antibody with a cell sample to beanalyzed; (iii) incubating the resultant of step (ii) with a detectingantibody which is capable of binding specifically to the BLT2 proteinand conjugated with a label generating a detectable signal; and (iv)detecting the signal generated from the label conjugated with thedetecting antibody.

The detecting antibody has a label generating a detectable signal. Thelabel includes, but not limited to, a chemical (e.g., biotin), anenzymatic (e.g., alkaline phosphatase, horseradish peroxidase,(3-galactosidase and Cytochrome P450), a radioactive (e.g., C14, 1-125,P32 and S35), a fluorescent (e.g., fluorescein), a luminescent, achemiluminescent and a FRET (fluorescence resonance energy transfer)label. Various labels and methods for labeling antibodies are well knownin the art (Ed Harlow and David Lane, Using Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1999).

The detection of the signal generated from the label conjugated with thedetecting antibody can be carried out by various processes well known inthe art. The detection of the signal enables to analyze the BLT2 proteinin a quantitative or qualitative manner. Where biotin and luciferase areused as labels, the signal detection may be achieved by use ofstreptavidin and luciferin, respectively.

The measurement of signal intensities generated from the immunoassaydescribed above is indicative of asthma. When the signal to the BLT2protein in a biosample to be diagnosed is measured to be higher thannormal samples, the biosample can be determined to have asthma. The kitof the present invention may optionally include other reagents alongwith primers, probes or antibodies described above. For instance, wherethe present kit may be used for nucleic acid amplification, it mayoptionally include the reagents required for performing PCR reactionssuch as buffers, DNA polymerase (thermostable DNA polymerase obtainedfrom Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermis flavus, Thermococcus literalis, and Pyrococcusfuriosus (Pfu)), DNA polymerase cofactors, anddeoxyribonucleotide-5-triphosphates. The kits, typically, are adapted tocontain in separate packaging or compartments the constituentsafore-described.

The kits for detecting or diagnosing asthma permit to determine thedevelopment, aggravation and alleviation of asthma. In this regard, theterm used herein “detecting or diagnosing” with reference to diseasemeans not only the determination of the existence of disease but alsothe development, aggravation and alleviation of disease.

The pharmaceutically acceptable carrier contained in the pharmaceuticalcomposition of the present invention, which is commonly used inpharmaceutical formulations, but is not limited to, includes lactose,dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassiumphosphate, arginate, gelatin, potassium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrups,methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc,magnesium stearate, and mineral oils. The pharmaceutical compositionaccording to the present invention may further include a lubricant, ahumectant, a sweetener, a flavoring agent, an emulsifier, a suspendingagent, and a preservative. Details of suitable pharmaceuticallyacceptable carriers and formulations can be found in Remington'sPharmaceutical Sciences (19th ed., 1995), which is incorporated hereinby reference.

According to another aspect of the present invention, there is provideda kit for detecting asthma, which comprises a primer or probe having anucleotide sequence complementary to the nucleotide sequence of the Racgene. Therefore, any methodology or kit developed based on theinformation that Rac overexpression is detected in the lung airway of apatient with asthma may be included in the present invention.

According to another aspect of the present invention, there is provideda kit for detecting asthma, which comprises an antibody bindingspecifically to Rac protein. The diagnosing kit for asthma may beconstructed by incorporating an antibody binding specifically to the Racprotein. A pharmaceutical composition of this invention may beadministered orally or parenterally (e.g., intravenous injection,subcutaneous injection, intramuscular injection and local injection).

The administration of a compound or a combination of compounds for thetreatment of asthma may be by any suitable means that results in aconcentration of the therapeutic that, combined with other components,is effective in ameliorating, reducing, or stabilizing a neoplasia. Thecompound may be contained in any appropriate amount in any suitablecarrier substance, and is generally present in an amount of 1-95% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for parenteral (e.g.,subcutaneously, intravenously, intramuscularly, or intraperitoneally)administration route.

The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

The term “therapeutically effective amount” as used herein means anamount of the substance that is capable of producing a medicallydesirable result in a treated subject. The correct dosage of thepharmaceutical compositions of this invention will be varied accordingto the particular formulation, the mode of application, age, body weightand sex of the patient, diet, time of administration, condition of thepatient, drug combinations, reaction sensitivities and severity of thedisease. According to a preferred embodiment of this invention, a dailysuitable dosage unit for human host ranges from 0.001-100 mg/kg (bodyweight). Human dosage amounts can initially be determined byextrapolating from the amount of compound used in mice, as a skilledartisan recognizes it is routine in the art to modify the dosage forhumans compared to animal models. In certain embodiments it isenvisioned that the dosage may vary from between about 1 lig compound/Kgbody weight to about 5000 mg compound/Kg body weight; or from about 5mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kgbody weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg bodyweight to about 2000 mg/Kg body weight; or from about 100 mg/Kg bodyweight to about 1000 mg/Kg body weight; or from about 150 mg/Kg bodyweight to about 500 mg/Kg body weight. In other embodiments this dosemay be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, mg/Kg body weight. In other embodiments, it is envisaged that dosesmay be in the range of about 5 mg compound/Kg body to about 20 mgcompound/Kg body. In other embodiments the doses may be about 8, 10, 12,14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may beadjusted upward or downward, as is routinely done in such treatmentprotocols, depending on the results of the initial clinical trials andthe needs of a particular patient.

According to the conventional techniques known to those skilled in theart, the pharmaceutical compositions of this invention can be formulatedwith pharmaceutical acceptable carrier and/or vehicle as describedabove, finally providing several forms including a unit dosage form.Non-limiting examples of the formulations include, but not limited to, asolution, a suspension or an emulsion, an extract, an elixir, a powder,a granule, a tablet, a capsule, emplastra, a liniment, a lotion and anointment.

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or incontact with the thymus; (v) formulations that allow for convenientdosing, such that doses are administered, for example, once every one ortwo weeks; and (vi) formulations that target a neoplasia by usingcarriers or chemical derivatives to deliver the therapeutic agent to aparticular cell type (e.g., neoplastic cell). For some applications,controlled release formulations obviate the need for frequent dosingduring the day to sustain the plasma level at a therapeutic level. Anyof a number of strategies can be pursued in order to obtain controlledrelease in which the rate of release outweighs the rate of metabolism ofthe compound in question. In one example, controlled release is obtainedby appropriate selection of various formulation parameters andingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the therapeutic is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra. Compositions for parenteral use may be provided in unitdosage forms (e.g., in single-dose ampoules), or in vials containingseveral doses and in which a suitable preservative may be added (seebelow). The composition may be in the form of a solution, a suspension,an emulsion, an infusion device, or a delivery device for implantation,or it may be presented as a dry powder to be reconstituted with water oranother suitable vehicle before use. Apart from the active agent thatreduces or ameliorates a neoplasia, the composition may include suitableparenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease.

Furthermore, the composition may include suspending, solubilizing,stabilizing, pH-adjusting agents, tonicity adjusting agents, and/ordispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active antineoplastic therapeutic(s)are dissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol or thelike. Asthma is a chronic inflammatory disease of the airwaycharacterized by eosinophil infiltration, mucus hypersecretion and AHR.Although there have been many studies of the role of BLT1 in asthma, therole of BLT2 has not yet been defined. By employing a pharmacologicalBLT2 antagonist and an antisense nucleotide sequence that blockedendogenous BLT2 expression, BLT2 was demonstrated to play a criticalrole in the development of AHR and airway inflammation. Results showedthat BLT2 mediates the asthmatic response by stimulating ROS generationand subsequent NF-KB activation. This is the first report that BLT2 isinduced by allergenic stimuli and that blockade of BLT2 mitigates theasthmatic response.

A number of earlier studies have shown that the ‘LTB4-BLT1’ pathwayplays a central role in the early chemoattraction of granulocytes suchas eosinophils to inflamed tissues, thereby acting as a localinflammatory mediator (3, 35). The LTB4-BLT1 pathway is also consideredpivotal for the allergen-mediated recruitment of effector CD4+ and CD8+T lymphocytes into airways, thereby controlling the immunologicalresponse, as well (10-12, 36). LTB4-BLT1 is therefore believed tocontribute to the development of asthma through recruitment ofgranulocytes and effector CD4+ and CD8+ T lymphocytes. In contrast toBLT1, BLT2 has a low affinity for LTB4, and no clear physiologicalfunction has yet been identified for it. Surprisingly and unexpectedly,BLT2 was dramatically upregulated during OVA-induced allergic pulmonaryinflammation in the asthma model. Unlike BLT1, which is mainly expressedin myeloid leukocytes and specific classes of T cells, BLT2 was inducedin the airway epithelium and in parts of the endothelium (FIG. 1C),where LTB₄ is abundantly generated in response to allergen challenge.After aerosol challenge with OVA, airway mast cells and alveolarmacrophages are activated. These cells are thought to be the majorsource of LTB₄ in the airways at early times following allergenchallenge. Although LTB₄ is believed to attract eosinophils, neutrophilsand differentiated T cells to airways via the BLT1 they express, LTB₄may also interact with BLT2 induced in the airway epithelium in thelocal microenvironment and stimulate intracellular signaling leading toupregulation of VCAM-1 and other proinflammatory proteins. The inducedVCAM-1 could in turn enhance trafficking of inflammatory leukocytesacross the epithelial layer to the airways, thereby contributing to adevelopment of airway inflammation and AHR. In fact, leukocyteemigration into the alveolar compartments is a prominent feature ofacute and chronic inflammatory lung injuries such as asthma (37). Duringthis emigration process, airway epithelial cells are probably importantnot only for retention and activation of leukocytes, but also forregulating their passage into the airways. In any event, thedistribution and function of ‘LTB₄-BLT2’ during the asthmatic responseappear to be unique and distinguishable from that of LTB₄-BLT1. BLT2clearly plays only a minimal role in T cell recruitment; consequently, Tcell recruitment to airways remained intact after administration ofantisense BLT2 (FIGS. 8A and 8B), while AHR and airway inflammation wereattenuated. This means the mechanism by which BLT2 inhibition suppressesAHR and airway inflammation is independent of T cell recruitment. On theother hand, no expression of BLT2 was detected in CD4+ T cells and CD8+TEFF cells, though they strongly express BLT1 (10, 38).

Without being bound to a particular theory, ROS is a major downstreamcomponent of the LTB4-BLT2 pathway mediating AHR and airway inflammationin allergic asthma. Accumulating evidence suggests that ROS and theoxidative stress they cause play crucial roles in the pathogenesis ofairway inflammation and AHR (29, 39-41). At later stages, moreover, theinflammatory cells recruited to the asthmatic airways have thecapability to produce ROS, and the ROS released by eosinophils and otherleukocytes infiltrating the airways cause the tissue injury observed inasthma (42).

That said, in this case it is not believed that ROS are acting merely asnonspecific pathogenic mediators of oxidative stress. Instead, theyappear to have a specific signaling function in the pathway leading toupregulation of target genes associated with asthma. In support of thisidea, BLT2 activity was previously shown to leads to enhanced ROSgeneration, which in turn mediates specific intracellular signalingresponses (7, 37). Although the molecular basis of ROS-mediatedinduction of AHR and inflammation remains unknown, recent studies haveshown that ROS generation in asthma leads to activation of theredox-sensitive transcription factor NF-KB (32, 43), which is present inmost cell types and plays a critical role in immune and inflammatoryresponses, including asthma (44). For instance, NF-KB activationcontributes to the development and maintenance of asthma in thebronchial epithelium (45), and has been observed in airway epithelialcells (46) in which BLT2 mRNA is induced during the OVA-induced allergicresponse. Consistent with those observations, in the present study NF-KBlevels were substantially elevated in extracts of lung tissue from micewith OVA-induced asthma and were specifically suppressed by BLT2antisense (FIG. 6A). It is known that activation of NF-KB induces avariety of proinflammatory genes, including adhesion molecules (e.g.,VCAM-1) (33, 34). As expected, expression of VCAM-1 increased followingallergen challenge, and BLT2 antisense or a receptor antagonist reducedits expression (FIGS. 6C and 6E), whereas BLT1 antisense had no effect(data not shown).

BLT2−/− knockout mice are difficult to generate because the BLT2 generesides within the BLT1 locus (47). Disruption of BLT2 thereforeinterferes with BLT1 expression, making it difficult to interpret theoutcome. Therefore, transgenic mice were prepared overexpressing BLT2and detected elevated levels of ROS in their BAL fluid (Cho et al.,unpublished observation). In addition, significant induction of AHR wasobserved, even before OVA challenge, which further supports the proposedmediatory role of BLT2 in the pathogenesis of asthma (Cho et al.,unpublished observation).

Applicants have discovered a relationship between asthma and aBLT2-linked signaling cascade. Without being bound to a particulartheory, LTB4 exerts its effects through both BLT1- and BLT2-dependentsignaling pathways and that the two may cooperate during the developmentof allergic asthma, although attenuation of either pathway suppressedasthmatic symptoms. A better understanding of the BLT2-linked pathwayand possible cross-regulation between the BLT1 and BLT2 pathways shouldhelp to clarify their role in LTB₄-mediated allergic pathogenesis. Thefinding that a LTB₄-BLT2-ROS pathway is involved in asthma could serveas the b for the development of new diagnostic tools and treatments forallergic disease.

Practical and presently preferred embodiments of the present inventionare illustrated as shown in the following Examples. However, it will beappreciated that those skilled in the art, on consideration of thisdisclosure, may make modifications and improvements within the spiritand scope of the present invention.

EXAMPLES Example 1 Sensitization and Challenge of Mice

Female BALB/c mice and C57BL/6 mice (7 weeks old; 18-20 g) were obtainedfrom Orientbion Inc. (Seoungnam, Korea). Sensitization and challengewere carried out as described previously with some modification (28).Briefly, female C57BL/6 mice (7 weeks old; 18-20 g) were immunized byintraperitoneal (i.p.) injection of 2001.ig ovalbumin (OVA) emulsifiedin 2.5 mg of adjuvant aluminum hydroperoxide gel (alum) (Pierce,Rockford, Ill.). A second i.p. injection of 20 i.ig OVA adsorbed ontoalum (2.5 mg) was administered 10 days later. After an additional 10days, mice were exposed to an aerosol of 1% OVA in saline for 30 mindaily on 3 consecutive days. On day 25, mice were finally challenged byprovocation with 10% OVA aerosol. For inhibition experiments, sense orantisense BLT2 (1.6 mg/kg) was injected intravenously 24 h and then 1 hbefore the 10% OVA challenge. The mice were then killed on day 27 toassess asthmatic phenotypes. Antisense BLT2 oligonucleotide(5′-GCTCAGTAGTGTCTCATTCC-3′ (SEQ ID NO: 15)), sense BLT2 oligonucleotide(5′-GGAATGAGACACTACTGAGC-3′ (SEQ ID NO: 16)).

Alternatively, BALB/c mice were sensitized on day 1 by i.p. injection of20 i.ig OVA emulsified in 2.5 mg of alum (Pierce, Rockford, Ill.),followed by an identical booster injection administered on day 14. Ondays 21, 22 and 23 after initial sensitization, the mice were challengedfor 30 min with an aerosol of 1% OVA using an ultrasonic nebulizer.LY255283 (2.5 mg/kg) or vehicle control (DMSO) was administeredintravenously 1 h before 1% OVA challenge. Mice were killed on day 25,to assess asthmatic phenotypes. 2′,7′-dichorofluorescein diacetate(DCF-DA) was purchased from Molecular Probes (Eugene, Oreg.). BSA andDMSO were from Sigma-Aldrich (St. Louis, Mo.). Acetyl-methacholinechloride was purchased from Sigma-Aldrich (St. Louis, Mo.). All otherchemicals were from standard sources and were of molecular biology gradeor higher. All mice were maintained and bred under specificpathogen-free conditions in the Korea University mouse facility, andexperiments were conducted within the parameters of an approved protocolby the Animal Research Committee.

Example 2 Induction of BLT2 mRNA in the OVA-Induced Asthmatic Mouse Lung

1) Quantification of LTB₄ (FIG. 1A)

Levels of LTB₄ were quantified with the leukotriene B4 enzymeimmunoassay (EIA) Biotrak™ system (Amersham Biosciences, UK). Briefly,200 i.il BAL fluid were concentrated by freeze-drying for 12 h andreconstituted in assay buffer. The sensitivity of the assay was 0.3pg/well, which is equivalent to 6 pg/ml.

2) Semiquantitative RT-PCR (FIG. 1B)

Total RNA was extracted from lung samples using Easy-blue RNA extractionreagent (Intron, Korea). The extracted RNA (1 i_tg) was reversetranscribed for 1 hr at 42° C. and amplified by PCR using the followingprimers: for mouse BLT2, 5′-CAGCATGTACGCCAGCGTGC-3′ (sense; SEQ ID NO:17) and 5′-CGATGGCGCTCACCAGACG-3′ (antisense; SEQ ID NO: 18); and formouse BLT1, 5′-GCATGTCCCTGTCTCTGTTG-3′ (sense; SEQ ID NO: 19) and5′-CGGGCAAAGGCCTTAGTACG-3′ (antisense; SEQ ID NO: 20). For thesemiquantitative analysis of transcripts, the optimal PCR conditionswere first determined by linear amplification ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thereafter, 28 cycleswas used for BLT amplification and was found to be in the linear range.The amplified PCR products were separated by electrophoresis on 1.2%agarose gel and stained with ethidium bromide.

3) In Situ Hybridization for BLT2 in Mouse (FIG. 1C)

The cDNA encoding mouse BLT2 was amplified by PCR with the mouse BLT2primers and confirmed by sequencing. All linearized vectors weretranscribed with T7 RNA polymerase and digoxigenin (DIG) RNA labelingmix (Roche, Germany). Embedded mouse lung tissues were deparaffinizedwith xylene, after which in situ hybridization was carried out using anin situ hybridization detection kit (InnoGenex, CA) according to themanufacturer's protocol.

Example 3 Blockade of BLT2 Signaling Using a Pharmacological Antagonist(LY255283) Suppresses Airway Inflammation

1) Semiquantitative RT-PCR (FIG. 2A)

Total RNA was extracted from lung samples using Easy-blue RNA extractionreagent (Intron, Korea). The extracted RNA (1 i_tg) was reversetranscribed for 1 hr at 42° C. and amplified by PCR using the followingprimers: for mouse BLT2, 5′-CAGCATGTACGCCAGCGTGC-3′ (sense; SEQ ID NO:17) and 5′-CGATGGCGCTCACCAGACG-3′ (antisense; SEQ ID NO: 18); and formouse BLT1, 5′-GCATGTCCCTGTCTCTGTTG-3′ (sense; SEQ ID NO: 19) and5′-CGGGCAAAGGCCTTAGTACG-3′ (antisense; SEQ ID NO: 20). For thesemiquantitative analysis of transcripts, the optimal PCR conditionswere first determined by linear amplification ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thereafter, 28 cycleswas used for BLT amplification and was found to be in the linear range.The amplified PCR products were separated by electrophoresis on 1.2%agarose gel and stained with ethidium bromide.

2) Real-Time PCR (FIG. 2B)

For real-time PCR, total RNA were extracted from lung tissue usingEasy-blue RNA extraction reagent (Intron, Korea), after which theextracted RNA was reverse transcribed using M-MLV reverse transcriptase(Invitrogen, CA). The PCR reactions were then carried out usingLightCycler 480 SYBR Green I Master (Roche, Germany) according to themanufacturer's 30 instructions.

3) BAL and Histological Analysis of Lung (FIGS. 2C and 2D)

Inflammatory cells in the BAL fluid were collected by centrifugation(1,000 g for 3 min) and washed once in PBS. Cells were counted using ahemocytometer, and viability was assessed by trypan blue exclusion. Inaddition, cytospin was carried out for each BAL sample, which was thenstained with Diff-Quick (Merck, Dorset, U.K.), enabling differentialcell counts to be made. For histological analysis, the lungs of the micewere dissected 48 h after OVA challenge and fixed with 10% formaldehydein PBS, dehydrated in ethanol-xylene, and embedded in paraffin. Multipleparaffin-embedded 6-1.tm sections were placed on 0.5% gelatin-coatedslides, deparaffinized, and stained with hematoxylin-eosin (HE). Imageswere acquired using a BX51 microscope (Olympus, Tokyo, Japan) equippedwith a DP71 digital camera (Olympus, Tokyo, Japan).

Example 4 Blockade of BLT2 Signaling Using a Pharmacological AntisenseBLT2 Suppresses Airway Inflammation

1) Semiquantitative RT-PCR (FIG. 3A)

Total RNA was extracted from lung samples using Easy-blue RNA extractionreagent (Intron, Korea). The extracted RNA (1 i_tg) was reversetranscribed for 1 h at 42° C. and amplified by PCR using the followingprimers: for mouse BLT2, 5′-CAGCATGTACGCCAGCGTGC-3′ (sense; SEQ ID NO:17) and 5′-CGATGGCGCTCACCAGACG-3′ (antisense; SEQ ID NO: 18); and formouse BLT1, 5′-GCATGTCCCTGTCTCTGTTG-3′ (sense; SEQ ID NO: 19) and5′-CGGGCAAAGGCCTTAGTACG-3′ (antisense; SEQ ID NO: 20). For thesemiquantitative analysis of transcripts, the optimal PCR conditionswere first determined by linear amplification ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thereafter, 28 cycleswas used for BLT amplification and was found to be in the linear range.The amplified PCR products were separated by electrophoresis on 1.2%agarose gel and stained with ethidium bromide.

2) Real-Time PCR (FIG. 3B)

For real-time PCR, total RNA were extracted from lung tissue usingEasy-blue RNA extraction reagent (Intron, Korea), after which theextracted RNA was reverse transcribed using M-MLV reverse transcriptase(Invitrogen, CA). The PCR reactions were then carried out usingLightCycler 480 SYBR Green I Master (Roche, Germany) according to themanufacturer's instructions.

3) BAL and Histological Analysis of Lung (FIGS. 3C and 3D)

Inflammatory cells in the BAL fluid were collected by centrifugation(1,000 g for 3 min) and washed once in PBS. Cells were counted using ahemocytometer, and viability was assessed by trypan blue exclusion. Inaddition, cytospin was carried out for each BAL sample, which was thenstained with Diff-Quick (Merck, Dorset, U.K.), enabling differentialcell counts to be made. For histological analysis, the lungs of the micewere dissected 48 h after OVA challenge and fixed with 10% formaldehydein PBS, dehydrated in ethanol-xylene, and embedded in paraffin. Multipleparaffin-embedded 6-1.tm sections were placed on 0.5% gelatin-coatedslides, deparaffinized, and stained with hematoxylin-eosin (HE). Imageswere acquired using a BX51 microscope (Olympus, Tokyo, Japan) equippedwith a DP71 digital camera (Olympus, Tokyo, Japan).

Example 5 Effect of BLT2 Inhibition on Airway AHR

1) Determination of AHR in Response to Methacholine (FIGS. 4A and 4B)

Airway AHR was measured in unrestrained, conscious mice 24 h after thefinal OVA challenge using a whole-body plethysmograph, as previouslydescribed (29). Mice were placed in a barometric plethysmographicchamber (All Medicus Co., Seoul, Korea), and baseline readings weretaken and averaged for 3 min. Aerosolized methacholine in increasingconcentrations (from 6.25 mg/m 1-50 mg/ml) was nebulized through aninlet of the main chamber for 3 min. Readings were taken and averagedfor 3 min after each nebulization, and enhanced pause (Penh) wasdetermined. Signals were analyzed to derive whole body flow parametersincluding respiratory rate, tidal volume, inspiratory time (Ti),expiratory time (Te)i. peak inspiratory flow (PIF), peak expiratory flow(PEF), and relaxation time (RT). These parameters were used to calculateenhanced pause (Penh), a unitless parameter that is used as a measure ofairway responsiveness to methacholine. Penh reflects changes inpulmonary resistance during bronchoconstriction according to thefollowing equation: Penh=[(Te-RT)+RT]×(PEF-PIF).

Baseline Penh measurements for each animal were recorded for 3 min andaveraged. Results are expressed as the percentage increase of Penhfollowing challenge with each concentration of methacholine, where thebaseline Penh (after saline challenge) is expressed as 100%. Penh valuesaveraged for 3 min after each nebulization were evaluated.

Example 6 Attenuation of ROS Generation by BLT2 Inhibition

1) Measurement of ROS Levels in BAL Fluid (FIG. 5A)

ROS levels in BAL fluids were measured as a function of DCF fluorescenceas described previously (30). Briefly, cells in the BAL fluid werecollected by centrifugation (1,000 g for 3 min) and the pelleted cellswere washed with PBS and incubated for 10 min with the H202-sensitivefluorophore 2′,7′-dichorofluorescein diacetate (DCF-DA, 101AM)(Molecular Probes, Eugene, Oreg.), which, when taken up, fluorescentlylabels intracellular H202 with DCF. Following washing, the cells wereimmediately observed using a FACS Calibur™ (Becton Dickinson, FranklinLakes, N.J.). DCF fluorescence was excited at 488 nm and the evokedemission was filtered with a 515-nm long-pass filter.

2) Quantification of LTB4 (FIG. 5B)

Levels of LTB4 were quantified with the leukotriene B4 enzymeimmunoassay (EIA) Biotrak™ system (Amersham Biosciences, UK). In brief,200 i.il BAL fluid was concentrated by freeze-drying for 12 h andreconstituted in assay buffer. The assay was calibrated with standardLTB₄ ranging from 0.31 to 40 pg/well. Samples of BAL fluid and standardLTB₄ in 96-well plates were incubated with antiserum for 2 hr, followedby LTB₄ peroxidase conjugate for 1 h at room temperature. To removeunbound ligand, the wells were aspirated and washed 4 times with buffer.Substrate (tetramethylbenzidine) was then added, and the reaction wasstopped by adding an acid solution and the color read at 450 nm in aspectrophotometer. The sensitivity of the assay was 0.3 pg/well, whichis equivalent to 6 pg/ml. Statistical significance of differencesbetween groups was assessed by analysis of variance, and P<0.05 wasconsidered significant.

Example 7 Attenuation of NF-KB Activation by BLT2 Inhibition

1) Electrophoretic Mobility Shift Assay (EMSA) (FIG. 6A)

A double-strand oligonucleotide corresponding to the consensus NF-KBbinding motif and a mutant sequence were purchased from Santa CruzBiotechnology Inc. (Santa Cruz, Calif.) and labeled with y-32P-ATP usingT4 polynucleotide kinase (Roche, Germany). Labeled oligonucleotide wasthen separated from free y-32P-ATP on ProbeQuart™ G-50 microcolumns(Amersham Pharmacia Biotech, Ltd., UK) according to the manufacturer'sprotocol. Labeled oligonucleotide, 10 μg of nuclear extract and EMSAbuffer were incubated for 1 h at room temperature in a final volume of20 IA after which the reaction mixture was subjected to electrophoresis.

2) SDS-PAGE and Immunoblot Analysis of Lung Cell Lysates (FIGS. 6B-6E)

Lung samples were prepared as described for preparation of totalprotein. The cell lysates were centrifuged at 13,000 g for 10 min andthe supernatants subjected to SDS-PAGE on 10% acrylamide gels, followedby transfer to polyvinylidene difluoride (PVDF) membranes with a Novexwet transfer system (for 2 h at 100 V). The membranes were blocked for 1h in Tris-buffered saline (TBS) containing 0.05% (v/v) Tween 20 plus 5%(w/v) nonfat dry skim milk, and then incubated for 2 h with the primaryantibody in TBS containing 0.05% (v/v) Tween 20 plus 3% (w/v) BSA,followed for 1 h with horseradish peroxidase (HRP)-conjugated secondaryantibody before development by enhanced chemiluminescence (ECL)(Amersham Pharmacia Biotech, Ltd., UK).

Example 8 Enhanced Expression of BLT2 in Samples from ClinicallyAsthmatic Subjects

1) Bronchoscopy (FIG. 7)

Bronchial biopsy specimens were obtained from 4 nonasthmatic controls(normal), 4 mild bronchial asthma patients and 5 moderate bronchialasthma patients. The patients studied were recruited from the outpatientclinic of Soonchunhyang University Hospital, Korea. The subjects in thenonasthmatic control group had no history of broncho-pulmonary diseaseand had an FEV1>80% of predicted and an FEV1/FVC %>70%. The mildbronchial asthmatic group had an FEV1>70% and moderate bronchialasthmatic group had an FEV1<70%. All specimens were formalin-fixed,paraffin-embedded and processed for routine histological diagnosis. Thestudy was approved by the ethics committee of Soonchunhyang UniversityHospital, and the patients provided written informed consent. Thepattern of BLT2 expression in the bronchial biopsy specimens wasdetected immunohistochemically using an alkaline phosphatase substratesystem.

Example 9 BL T2 Inhibition does not Interfere with Recruitment of TLymphocytes into Airways

1) Flow Cytometric Analysis of Cells in BAL Fluid (FIGS. 8A-8B)

For flow cytometric analysis, the cells in the BAL fluid were suspendedin 501,t1 of PBS containing 0.01% sodium azide and 0.1% BSA. BALleukocytes were incubated for 30 min with 2.4G2 anti-Fcylll/ll receptor(BD PharMingen) and stained for 30 min at 4° C. with FITC-conjugatedanti-mouse TCR chain (BD PharMingen) and PE-cy5 anti-mouse CD8a (BDPharMingen) or PE rat anti-mouse CD4 (BD PharMingen). Cytofluorimetrywas performed with a FACS Caiibur™ (Becton Dickinson, Franklin Lakes,N.J.), and the results were analyzed with CellQuest software(Becton-Dickinson).

Example 10 Antisense Rac Oligonucleotide Experiment

Oligonucleotides, from Genotech Co. (Korea), were synthesized with aphosphorothioate backbone to improve the resistance to endonuclease. Theantisense oligonucleotide consisted of 17 nucleotides analogues to the5′ end of the murine Rac mRNA sequence, which spans the translationinitiation site. The control Rac oligonucleotide contained the samenucleotide composition as the antisense oligonucleotide.Oligonucleotides (1.25 mg/kg of weight) were injected into the tailveins of the mice 24 and 4 hr before OVA provocation. The sequences ofthe oligonucleotides used in this study were as follows. Control Rac:5′-GATCAGTGCACACAGTG-3′ (SEQ ID NO: 21); Antisense Rac:5′-CACTTGATGGCCTGCAT-3′ (SEQ ID NO: 22).

1) BAL and Histological Analysis of Lung (FIG. 9A)

Inflammatory cells in the BAL fluid were collected by centrifugation(1,000 g for 3 min) and washed once in PBS. Cells were counted using ahemocytometer, and viability was assessed by trypan blue exclusion. Inaddition, cytospin was carried out for each BAL sample, which was thenstained with Diff-Quick (Merck, Dorset, U.K.), enabling differentialcell counts to be made. For histological analysis, the lungs of the micewere dissected 48 hr after OVA challenge and fixed with 10% formaldehydein PBS, dehydrated in ethanol-xylene, and embedded in paraffin.

Multiple paraffin-embedded 6-1.tm sections were placed on 0.5%gelatin-coated slides, deparaffinized, and stained withhematoxylin-eosin (HE). Images were acquired using a BX51 microscope(Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus,Tokyo, Japan).

2) Electrophoretic Mobility Shift Assay (EMSA) (FIG. 9B)

A double-strand oligonucleotide corresponding to the consensus NF-KBbinding motif and a mutant sequence were purchased from Santa CruzBiotechnology Inc. (Santa Cruz, Calif.) and labeled with y-32P-ATP usingT4 polynucleotide kinase (Roche, Germany). Labeled oligonucleotide wasthen separated from free y-32P-ATP on ProbeQuart™ G-50 microcolumns(Amersham Pharmacia Biotech, Ltd., UK) according to the manufacturer'sprotocol. Labeled oligonucleotide, 10 μg of nuclear extract and EMSAbuffer were incubated for 1 h at room temperature in a final volume of20 IA after which the reaction mixture was subjected to electrophoresis.

3) SDS-PAGE and Immunoblot Analysis of Lung Cell Lysates (FIG. 9C)

Lung samples were prepared as described for preparation of totalprotein. The cell lysates were centrifuged at 13,000 g for 10 min andthe supernatants subjected to SDS-PAGE on 10% acrylamide gels, followedby transfer to polyvinylidene difluoride (PVDF) membranes with a Novexwet transfer system (for 2 h at 100 V).

The membranes were blocked for 1 h in Tris-buffered saline (TBS)containing 0.05% (v/v) Tween 20 plus 5% (w/v) nonfat dry skim milk, andthen incubated for 2 h with the primary antibody in TBS containing 0.05%(v/v) Tween 20 plus 3% (w/v) BSA, followed for 1 h with horseradishperoxidase (HRP)-conjugated secondary antibody before development byenhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Ltd., UK).

Result 1: Induction of BLT2 mRNA in the OVA-Induced Asthmatic Mouse Lung

To assess the role of BLT2 in OVA-induced allergic asthma, levels ofBLT2 and its ligand LTB4 were measured. As shown in FIG. 1A, LTB4 levelsin BAL fluid increased following OVA challenge in this murine model ofasthma, and peaked 48 h after the challenge. Semiquantitative RT-PCRanalysis showed that levels of BLT2 mRNA in the lung increaseddramatically after OVA challenge, while levels of BLT1 mRNA increasedonly slightly (FIG. 1B). To determine the distribution of BLT2expression in the lung, in situ hybridization with an antisense BLT2probe were next carried out and substantial elevation of BLT2 expressionwas detected in the epithelium with some induction in the endothelium(FIG. 1C). Thus, both LTB4 and its receptor BLT2 appear to beupregulated in asthma.

Result 2: Blockade of BLT2 Signaling Using a Pharmacological Antagonistor Antisense BLT2 Suppresses Airway Inflammation

To assess the possible mediatory role of BLT2 in asthmatic airwayinflammation, a specific BLT2 antagonist, LY255283, was administratedintravenously 1 hr before the 1% OVA challenge, and the mice were thensacrificed 48 h after challenge. Semiquantitative RT-PCR (FIG. 2A) orreal-time PCR analysis (FIG. 2B) showed that the level of BLT2 mRNA inthe lung was greatly elevated after the OVA challenge, andadministration of LY255283 significantly reduced BLT2 levels.Histological analysis of the infiltration of inflammatory cells into thelung revealed increased airway obstruction and leukocyte infiltrationfollowing OVA challenge (FIGS. 2C and 2D); again, LY255283 diminishedthe effect (FIG. 2C). Quantitative analysis of the histological samples,which entailed grading the airway inflammation as described in Materialsand Methods, revealed that LY255283 reduced the inflammation score by−66%, as compared to control (FIG. 2D).

To further evaluate the role of BLT2 in the pathogenesis of asthma, theeffect of antisense BLT2 was analyzed. In this experiment, antisenseBLT2 was administrated intravenously 24 hr and 1 hr before 10% OVAchallenge, and the mice were sacrificed 48 hr after challenge.Subsequent semiquantitative RT-PCR analysis showed that the antisenseBLT2 reduced BLT2 expression in the lung without interfering with thatof BLT1 (FIG. 3A). Similar results were also obtained with real-time PCRanalysis (FIG. 3B). Airway eosinophil accumulation is a hallmark ofasthmatic pulmonary inflammation, and accumulation of eosinophils wasdetected in BAL fluid, which peaked 48 hr after the 10% OVA challenge.Administration of antisense BLT2 reduced eosinophil infiltration in BALfluids by −87%, whereas sense BLT2 had no inhibitory effect (FIG. 3C).In addition, histological analysis revealed increased airway obstructionand leukocyte infiltration following OVA challenge, and this effect,too, was diminished by antisense BLT2 (FIG. 3D). Consistent with thosefindings, administration of antisense BLT2 reduced inflammation scoresby −67%, as compared to control.

Result 3: Effect of BLT2 Inhibition on Airway AHR

To examine the contribution of BLT2 to AHR, the increase in Penh(enhanced pause) was determined to be elicited by methacholine (6.25-50mg/ml). OVA-challenged mice developed significant AHR to the inhaledmethacholine, and LY255283 or antisense BLT2 dramatically reduced Penhby −70% (based on area-under-the-curve calculations), which suggeststhat BLT2 is in some way critical for the AHR reaction (FIGS. 4A-46).

Result 4: Attenuation of ROS Generation by BLT2 Inhibition

The ‘LTB4-BLT2’ cascade was previously shown to lead to enhanced ROSgeneration, which mediates various cellular effects (7). Therefore ROSlevels were measured in BAL fluid following OVA challenge. As expected,the ROS levels in BAL fluid increased in the OVA-challenged mice.Notably, injection of antisense BLT2, but not sense BLT2, dramaticallyreduced ROS levels by −70% (FIG. 5A), suggesting ROS act as a mediatorin the ‘LTB₄-BLT2’ signaling leading to asthmatic symptoms.Administration of antisense BLT2 suppressed the level of LTB₄ in BALfluid by −72% (FIG. 5B), suggesting there may be cross-talk between LTB₄and BLT2, such that each affects the other. Indeed, similar instances ofcrosstalk between eicosanoid lipid ligands and their receptors have beendescribed previously (30, 31).

Result 5: Attenuation of NF-KB Activation by BLT2 Inhibition

ROS were previously reported to affect redox-sensitive factors such asNF-KB and AP-1 (32). To investigate the downstream signaling mechanismby which ‘LTB4-BLT2’ causes asthmatic symptoms in vivo, EMSA was used toassess NF-KB activation in the lungs of OVA-challenged mice. Nuclearextracts from lung tissue were tested for their ability to bind a32P-labeled oligonucleotide corresponding to the NF-KB consensussequence. OVA challenge elicited an increase in NF-KB binding activity,which was attenuated by prior administration of antisense BLT2 (FIG.6A). Moreover, competition assays using excess unlabeled oligonucleotide(cold) confirmed that the binding was specific. NF-KB normally residesin the cytoplasm in an inactivated form complexed with 1 KB-a. Uponstimulation, 1 KB-a is rapidly phosphorylated and degraded, allowingNF-KB to translocate into the nucleus. As a further indication of NF-KBactivation, the level of IKB-a following BLT2 inhibition was analyzed.Substantial degradation of 1 KB-a following OVA challenge was detected,but antisense BLT2 (FIG. 6B) or LY255283 (FIG. 6D) reduced thatdegradation by −50%. It is known that activation of NF-KB induces avariety of inflammatory genes, including adhesion molecules (e.g.,VCAM-1) (33). Therefore the effect of BLT2 blockade on levels of VCAM-1,which is regulated by NF-KB and is reportedly involved in eosinophilinfiltration (34), was also examined.

As shown in FIGS. 6C AND 6E, OVA challenge caused induction of VCAM-1 inlung tissue, and antisense BLT2 or LY255283 suppressed this effect by−60%.

Result 6: Enhanced Expression of BLT2 in Samples from ClinicallyAsthmatic Subjects

Immunohistochemical analysis was used to determine whether BLT2 levelsare also elevated in human asthmatic subjects. Bronchial biopsyspecimens were obtained from nonasthmatic controls (n=4) and patientswith mild (n=4) or moderate (n=5) bronchial asthma. In accordance withthe results obtained with the murine model of asthma (FIG. 1C), BLT2expression was found to be significantly elevated in all mild andmoderate bronchial asthma specimens (FIG. 7). Representative bronchialspecimens from asthma patients (FIG. 7, all but left top panel) showhighly induced expression of BLT2, whereas those from healthy controlsdo not (FIG. 7, left top, middle and bottom panels), which indicates arole of BLT2 in the clinical pathogenesis of asthma. BLT2 expression wasmainly elevated in the airway epithelial layers and microvascularendothelium in patient lung samples, which is similar to the patternobserved in the murine model of asthma.

Result 7: BLT2 Inhibition does not Interfere with Recruitment of TLymphocytes into Airways

BLT1 was found to be responsible for early recruitment of CD4+ and CD8+T cells into the airways in a model of allergic pulmonary inflammation,suggesting that the LTB4-BLT1 pathway is involved in linking earlyimmune system activation and effector T cell recruitment (11). Thus,BLT2 might plays a similar role in T cell trafficking into airways.Significant numbers of CD4+ and CD8+ T cells were recruited into BALfluid 12 hr after OVA challenge (FIGS. 8A and 8B). Importantly,antisense BLT2 had no inhibitory effect on T cell trafficking intoairways, while injection of antisense BLT1 markedly diminished thisrecruitment of CD4+ and CD8+ T cells by −93% and −95%, respectively.This suggests that the actions mediated by ‘LTB4-BLT2’ are quitedistinct from those mediated by ‘LTB4-BLTT’ during the asthmaticresponse.

Result 8: Antisense Rac Inhibit Airway Inflammation

To examine the involvement of Rac in OVA-induced allergic inflammatoryresponses, the activation of Rac by OVA provocation in lung lysate wasexamined. It is well known that Rac translocates to the membrane fromthe cytosol when it is activated. Therefore, the membrane proteins fromthe lung tissues of OVA challenged mice was prepared, and the amount ofRac in the membrane fraction as a marker of Rac activation was compared.Rac was activated by OVA provocation in early time point (1 to 3 hr) andreturned to normal at 6 hr after provocation (FIG. 10A).

Because there is no specific inhibitor for Rac, antisense Racoligonucleotide was designed analogous to the 5′ end of murine Rac mRNAsequence, which spans the translation initiation site, to inhibit theendogenous expression of Rac. To confirm the effect of antisense Racoligonucleotides on the expression of endogenous Rac, theoligonucleotides were injected into the tail veins of the mice, whichwere sacrificed after 12 hr. As expected, antisense Rac oligonucleotideinhibited the endogenous expression of Rac, while the controloligonucleotide didn't show significant effect on the expression of Racin the lung tissues of the mice (FIG. 10B).

To assess the possible mediatory role of Rac in asthmatic airwayinflammation, a specific Rac antisense, was administrated intravenously24 hr and 1 hr before the 10% OVA challenge, and the mice were thensacrificed 48 hr after challenge. Histological analysis of theinfiltration of inflammatory cells into the lung revealed increasedairway obstruction and leukocyte infiltration following OVA challenge(FIG. 9A); again, Rac antisense diminished the effect. Nuclear extractsfrom lung tissue were tested for their ability to bind a ³²P-labeledoligonucleotide corresponding to the NF-KB consensus sequence. OVAchallenge elicited an increase in NF-KB binding activity, which wasattenuated by prior administration of antisense Rac (FIG. 9B). Moreover,competition assays using excess unlabeled oligonucleotide (cold)confirmed that the binding was specific.

It is known that activation of NF-KB induces a variety of inflammatorygenes, including adhesion molecules (e.g., VCAM-1) (33). Therefore theeffect of Rac blockade on levels of VCAM-1, which is regulated by NF-KBand is reportedly involved in eosinophil infiltration (34), was alsoexamined. As shown in FIG. 9C, OVA challenge caused induction of VCAM-1in lung tissue, and antisense Rac suppressed this effect (FIG. 9C).

Result 9: BLT2 Antisense Oligonucleotide Suppression Effect on BLT2Expression

Suppressed BLT2 expression level was determined by RT-PCR. Rat2-BI_T2stable cells were plated at a density of 5×104 cells/plate on 6 wellplates. After 24 hr, cells were transiently transfected with BLT2specific antisense and sense oligonucleotide plasmid with Lipofectaminreagent and then incubated in fresh DMEM supplemented with 10% FBS foran additional 24 h. After additional incubation, the transfected cellswere harvested for BLT2 transcripts analysis.

Total RNA was reverse-transcribed and PCR amplify were performed withBLT2 forward primer: 5′-tctcatcgggcatcacaggt-3′ (SEQ ID NO: 23) andreverse primer: 5′-ccaagctccacaccacgaag-3′ (SEQ ID NO: 24).Non-transfected Rat2-BLT2 stable cells cDNA was used the negativecontrol and GAPDH was shown as internal control. FIGS. 9A-9C show thesuppression effect of BLT2 antisense oligonucleotide on BLT2 expressionlevel by RT-PCR.

The result showed that the level of BLT2 mRNA was reduced by theantisense oligonucleotide, however the level of BLT2 mRNA was notaffected by the sense oligonucleotide.

Result 10: BLT2 siRNA Suppression Effect on BLT2 Expression

BLT2 siRNA expression effect on BLT2 expression was addressed byNorthern blotting.

CHO-BLT2 stable cells were plated at a density of 1×105 cells/plate on60-mm dish. After 24 hr, cells were transiently transfected with BLT2specific siRNA, targeting for 1705-1724 bp in NM_019839;5′-GAAGGATGTCGGTCTGCTA-3′ (SEQ ID NO: 25), with oligofectamin reagentand then incubated in fresh RPMI 1640 supplemented with 10% FBS for anadditional 24 h. after additional incubation, total RNA was performedNorthern blot with [³²P]-dCTP labeled BLT2 probe. Scramble RNA andnon-coding sequence BLT2 siRNA were used the negative control. A 110 bpPCR fragment was amplified with pcDNA3.1-BLT2 clone using the followingtwo primers, forward primer: 5′-cttctcatcgggcatcacag-3′ (SEQ ID NO: 26)and reverse primer: 5′-atccttctgggcctacaggt-3′ (SEQ ID NO: 27). Thisprobe was located mainly in the BLT2 coding region. Total RNA wasextracted with TRIzol reagent and then loaded the ten microgram totalRNA for 2 hr in MOPS containing agarose gel. After this step, the totalRNA was transferred the Hybond N^(±) membrane for overnight with 20×SSCbuffer. The membrane was hybridized with [³²P]-dCTP labeled BLT2 probein the hybridization buffer for 18 h at 68° C. And then, washed in0.1×SSC (0.1% SDS) for 1 h at 68° C. and subjected to autoradiography.FIGS. 10A-10B show the suppression effect of BLT2 siRNA on BLT2expression level by Northern blot. The result showed that the level ofBLT2 mRNA was reduced by the BLT2 siRNA (coding sequence), however thelevel of BLT2 mRNA was not affected by the BLT2 siRNA (non-codingsequence). As disclosed above, the present inventors investigated therole of BLT2 in the pathogenesis of asthma using a murine model anddemonstrated that BLT2 plays a critical role in the development of AHRand airway inflammation by employing BLT2 inhibitors, such as antisenseoligonucelotide. Therefore, the BLT 2 inhibitors according to thepresent invention can be effectively used as a therapeutic compositionfor treating asthma.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

Example 11 Long Form BLT2 is Involved in Chemotactic Signaling,Chemotactic Motility, Cell Growth, ERK Activation, and Release of Th2Cytokine IL-13

To investigate the roles of long form BLT2 (LF-BLT2) and short form BLT2(SF-BLT2), experiments were performed to examine their properties. InCHO cells transfected with LF-BLT2 and SF-BLT2 expression constructs(2[tg DNA), levels of mRNA expression were similar as determined byRT-PCR assay using GAPDH transcript levels as a control (FIG. 12A). TheRT-PCR was performed under the following conditions: BLT2: lab primer(melting temperature 69° C.) for 30 cycles and GAPDH (meltingtemperature 58° C.) for 22 cycles. Additionally, FACS analysis of theCHO cells transfected with LF-BLT2 and SF-BLT2 expression constructsindicated similar levels of protein expression (FIG. 12B). FACS analysiswas performed under the using non-permeabilization, anti-HA antibody(Roche), and anti-mouse FITC antibody.

CHO cells transfected with LF-BLT2 and SF-BLT2 expression constructswere examined for reactive oxygen species (ROS) generation andchemotactic motility in the presence of LTB4.

LF-BLT2 showed a significantly enhanced ROS generation in the presenceof LTB4 compared to SF-BLT2 (−25% more ROS generation; FIG. 13A).LF-BLT2 also showed a significantly enhanced chemotactic migration inthe presence of LTB4 compared to SF-BLT2 (−30% more chemotacticmobility; FIG. 13B). Thus, LF-BLT2 was observed to be more active orefficient than SF-BLT2 in mediating chemotactic signaling and motility.Rat-2 cells transfected with LF-BLT2 and SF-BLT2 were examined forgrowth and proliferation and ERK activity in the presence of LTB4.SF-BLT2 transfected cells showed about the same growth as LF-BLT2transfected cells, but, in the presence of LTB4, significantly enhancedgrowth was observed by LF-BLT2 compared to SF-BLT2 (FIG. 14A). SF-BLT2transfected cells showed about the same ERK activity as LF-BLT2, but, inthe presence of LTB₄, a significantly enhanced ERK activation wasobserved by LF-BLT2 compared to SF-BLT2 (FIG. 14B).

To examine the roles of long form BLT2 (LF-BLT2) and short form BLT2(SF-BLT2) in asthma, LF-BLT2 and SF-BLT2 expression constructs weretransfected into bone marrow-derived mast cells (BMMCs), which areinvolved in the asthmatic allergic response. LF-BLT2 and SF-BLT2 nucleicacid sequences were inserted into the pcDNA3.1-LF-BLT2 plasmid. BMMCwere transiently transfected with pcDNA3, pcDNA3-short form BLT2 orpcDNA3-long form BLT2 for 24 hr, and allowed to overexpress LF-BLT2 andSF-BLT2. BMMC were transfected using a MP-100 Microporator (Digital Bio,Seoul, Korea) according to the manufacturer's instructions. Briefly,1×10⁶ cells in 100 pi of resuspension buffer (Digital Bio) containingpcDNA3, pcDNA3-short form BLT2 or pcDNA3-long form BLT2 wereelectroporated using one pulse of 1,400 V for 30 ms. Cells were culturedin complete medium without antibiotics for 48 h, and the mRNA level ofBLT2 was then analyzed by RT-PCR. For cells exposed to LTB₄, cells wereincubated with 300 nM of LTB₄ for 1 hr and then harvested for detectionof IL-13 transcripts by semi-quantitative RT-PCR with specific primers.The data show that expression of IL-13, a critical asthma-associated Th2cytokine, was highly induced by transient transfection with long formBLT2-expression plasmid, and not by short form BLT2 plasmid in thepresence of LTB₄ (FIG. 15) The LTB₄-evoked IL-13 induction in mast cellsindicated that LF-BLT2 acted in an allergic response. Thus, in anasthmatic role, long form BLT2 demonstrated a distinguishable functionfrom short form BLT2 in BMMCs.

Example 12 Generation of Anti-Long Form BLT2 Antibody Having BLT2Neutralizing Activity

To study long form BLT2, a long form BLT2 antibody was generated using a14-mer peptide present on long form BLT2 (PTPERPLWRLPPTC; peptide Ab-1;N-terminal sequences 14-27 amino acid residues), and not short formBLT2. Mice were immunized intraperitoneally with peptide Ab-1(PTPERPLWRLPPTC) conjugated to BSA (peptide 2.0; 100 μg/CFA/head).

Mice were immunized every 3 weeks. Four days after the last booster,mice were sacrificed and spleen cells were collected.

Spleen cells were fused with SP2 cells (American Type CultureCollection) by using PEG 4000 (Boehringer) at a 1:1 ratio. The PEGmediated fusion were performed according to the procedures previouslydescribed by Harlow and Lane (Harlow, E.; Lane, D., Eds.; Antibodies, alaboratory manual; Cold Spring Harbor Laboratory: New York, 1988; pp139-243). Fused cells were distributed over 96 well tissue cultureplates at 2,000 cells per well in complete DMEM medium containing 100[t.M hypoxanthine, 0.4 [t.M aminopterin, and 16 [t.M thymidine (HAT;Sigma). Medium was replaced weekly.

For the identification of antigen-reacting mAbs, ELISA-based screeningwas performed.

Briefly, microtiter plates were coated at 4° C. overnight with 100μ1 perwell of peptide Ab-1 conjugated with BSA (peptide 2.0; 10 μg/ml) dilutedin PBS. Plates were blocked with blocking buffer (PBS containing 1% BSA(Sigma) and 0.05% Tween 20 (Sigma)) for Ihr at room temperature.Hybridoma supernatant (1000 per well) was transferred into the ELISAplates. Binding reaction was carried out at room temperature for 2 h.Subsequently, plates were washed four times with washing buffer (PBScontaining 0.05% Tween 20 (Sigma)), and 100μ1 of HRP-conjugated goatanti-human Fab (Sigma) diluted 1:10,000 in binding buffer (PBScontaining 1% BSA (Sigma)) was added, and reactions were carried out atroom temperature for Ihr. Finally, plates were washed four times, and100μ1 of TMB substrate (Kirkegaard & Perry Laboratories) per well wasadded. The absorbance was determined at 490 nm.

To analyze the level of BLT2 expression, 253J-BV bladder cancer cellswere fixed with 3% paraformaldehyde and permeabilized with 0.1% TritonX-100 in PBS. After being blocked with 2% BSA for 30 min, cells wereincubated with the primary BLT2 antibody. Cells were then washed 3 timeswith PBS and incubated with FITC-conjugated anti-rabbit IgG (Invitrogen,Carlsbad, Calif.). Cells (10,000 per sample) were then analyzed with aflow cytometer (FACSCalibur™) using Cell Quest software, as describedpreviously. Data are expressed as the mean fluorescence intensity. TheFACS results shown in FIG. 16A are representative of three independentexperiments with similar results.

Of 22 potential candidates, FACS analysis indicated at least 6LF-BLT2-recognizing positive antibodies were obtained (FIG. 16B).Positive antibodies included: BLT2-LF-38 (#9 in FIG. 16B), BLT2-LF-45(#10 in FIG. 16B) BLT2-LF-62-5 (#19 in FIG. 16B), BLT2-LF-26-22 (#20 inFIG. 16B), BLT2-LF-20 (#21 in FIG. 16B), BLT2-LF-12-3 (#22 in FIG. 16B).In addition to using or characterizing the 6 positive antibodies,antibody BLT2-LF-13 (#4 in FIG. 16B) which did not recognize LF-BLT2 wasused as a negative control.

To test whether anti-LF-BLT2 antibodies have selective inhibitoryactivity of BLT2 function and, thus, BLT2 neutralizing activity, theeffect of anti-LF-BLT2 antibody on the chemotaxis property of stablyexpressing BLT2 CHO cells, BLT2 transiently transfected CHO cells, andBLT1 transiently transfected CHO cells was examined. BLT2 expressingcells were prepared and maintained in the media with 0.5 mg/mL G418.Transiently transfected CHO cells (CHO-vector, CHO-BLT1, and CHO-BLT2)were transfected with pcDNA3.1, pcDNA3.1-long form BLT2 or pcDNA3.1-BLT1plasmid. Chemotactic motility was assayed using Transwell chambers with6.5-mm-diameter polycarbonate filters (8-1.tm pore size, CorningCostar), as previously described. Briefly, the lower surfaces of thefilters were coated with 10 i.tg/mL fibronectin in serum-free RPMI 1640medium for 1 hr at 37° C. Dry, coated filters containing various amountsof LTB₄ were placed in the lower wells of the Transwell chambers, afterwhich 100 μL of CHO cells stably expressing BLT2 or transientlyexpressing BLT1 and BLT2 in serum-free RPMI 1640 were loaded into thetop wells, yielding a final concentration of 2.5×10⁴ cells/mL. Whenassessing the effects of inhibitors, cells were pretreated with therespective inhibitor for 30 min before seeding. After incubation at 37°C. in 5% CO₂ for 3 hr, the filters were fixed for 3 min with methanoland stained for 10 min with hematoxylin and eosin. Chemotaxis wasquantified by counting the cells on the lower side of the filter underan optical microscope (magnification, ×200). Six fields were counted ineach assay; each sample was assayed in duplicate; and the assays wererepeated twice.

Chemotactic migration was dramatically inhibited by anti-LF-BLT2antibody (BLT2-LF-26-22; #20 in FIG. 16B) in both CHO-BLT2 stable cellsand BLT2-transiently transfected CHO cells (FIG. 17a ), but noinhibitory effect by control antibody (BLT2-LF-13) (#4 in FIG. 16B) wasdetected (FIG. 17b ). However, BLT2 neutralizing antibody did not showany inhibitory effect on the chemotactic migration caused by BLT1transfected CHO cells (FIG. 17c ), indicating that the inhibitory effectof anti-LF-BLT2-#26-22 (#20 in FIG. 16B) was specific to BLT2. Thus,anti-LF-BLT2 antibody display neutralizing activity specific for BLT2.

Example 13 Anti-Asthma Activities of Anti-Long Form BLT2 NeutralizingAntibody

LF-BLT2 IgG Ab were tested to determine if they hadanti-BLT2-neutralizing effect in a mouse model of asthma. Anti-LF-BLT2antibodies showed specific inhibitory activities on the asthmatic AHR(airway hyperresponsiveness) phenotype asthma-induced mouse (induced bymethacholine).

Female BALB/c mice and C57BL/6 mice (7 weeks old; 18-20 g) were obtainedfrom ORIENT BIO Inc. (Seoungnam, Korea). BALB/c mice were sensitized onday 1 by i.p. injection of 20 g OVA emulsified in 2.5 mg of alum(Pierce, Rockford, Ill.), followed by an identical booster injectionadministered on day 14. On days 21, 22 and 23 after initialsensitization, the mice were challenged for 30 min with an aerosol of 1%OVA using an ultrasonic nebulizer.

BLT2 IgM and IgG Ab (100 μg or 500 μg/mice) or control antibody (100μg/mice) was administered intravenously 1 hr before 1% OVA challenge.Mice were killed on day 25 to assess asthmatic phenotypes. Allexperimental animals used in this study were treated according toguidelines approved by the Institutional Animal Care and Use Committeeof Korea University.

AHR was measured in unrestrained, conscious mice 24 hr after the finalOVA challenge, using a whole-body plethysmograph as previouslydescribed. Aerosolized methacholine in increasing concentrations (from6.25 mg/ml-50 mg/ml) was nebulized through an inlet of the main chamberfor 3 min. Readings were taken and averaged for 3 min after eachnebulization, and the enhanced pause (Penh) was determined.

The effect of BLT2 inhibition on AHR in response to methacholine wasexamined in the OVA-challenged mice. OVA-challenged mice were pretreatedwith control antibody (100 μg/mice) and BLT2 IgG Ab (100 μg/mice, 50Oug/mice) 1 h before 1% OVA challenge and then analysis at 24 h after thelast OVA challenge. For each sample, 5 mice were used. Treatment withBLT2 IgG Ab at both doses used (100 μg/mice, 500 μg/mice) decreased Penhin response to methacholine compared to treatment with control antibody(FIG. 18A). The Penh of mice treated with BLT2 IgG Ab was similar tothat of normal mice.

BLT2 IgG Ab attenuated ROS generation (FIG. 18B). BALF was collected 48hr after last OVA challenge. OVA-challenged mice were pretreated withcontrol antibody (100 μg/mice), BLT2 IgG Ab (100 μg/mice, 500 μg/mice) 1hr before 1% OVA challenge and then sacrificed at 48 h after the lastOVA challenge. The cells present in the BALF were observed using aFACSCalibur™.

As disclosed above, the invention makes use of BLT2 inhibitors for (1)suppressing an allergic response (e.g., asthma), (2) suppressing immuneresponses of mast cells, (3) suppressing Th2 cytokine IL-13 release, and(4) suppressing one or more of eosinophil infiltration into lung airway,airway inflammation, and airway hyperresponsiveness. Also, thisinvention include (4) a novel strategy for screeing BLT2 signalinginhibitors by measuring the cell growth of Rat2-BLT2 stable cells. Thus,the invention provides strategies for targeting BLT2 overexpression orover-activation for developing therapeutic compositions against asthma.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991).

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

REFERENCES

The following documents are cited herein.

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The invention claimed is:
 1. A monoclonal antibody that specificallybinds to and inhibits expression or intracellular signaling of long-formBLT2 in a lung cell or immune cell.
 2. The monoclonal antibody of claim1, wherein the monoclonal antibody is used for the treatment of asthma.3. A pharmaceutical composition comprising the monoclonal antibody ofclaim 1 and a pharmaceutical carrier.
 4. A kit for the treatment ofasthma, the kit comprising the monoclonal antibody of claim
 1. 5. Thekit of claim 4, wherein the antibody selectively reduces the expressionor intracellular signaling of long-form BLT2 to the patient while theexpression or intracellular signaling of short-form BLT2 is notdisrupted.
 6. The kit of claim 4, wherein the antibody specificallybinds long-form BLT-2 in the region set forth by amino acids 1-31 of SEQID NO:
 3. 7. The kit of claim 4, wherein the antibody specifically bindslong-form BLT-2 in the region set forth by amino acids 14-27 of SEQ IDNO: 3.